Polynucleotide

ABSTRACT

The present invention relates to a polynucleotide comprising a ubiquitous chromatin opening element (UCOE) which is not derived from 13. The polynucleotide of any one of claims  1  to  7 , wherein the UCOE comprises the sequence of FIG.  20  between nucleotides 1 to 7627 or a functional homologue or fragment thereof. an LCR. The present invention also relates to a vector comprising the polynucleotide sequence, a host cell comprising the vector, use of the polynucleotide, vector or host cell in therapy and in an assay, and a method of identifying UCOEs. The UCOE opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional application of application Ser.No. 09/358,082, Filed Jul. 21, 1999, now U.S. Pat. No. 6,689,606, IssuedFeb. 10, 2004, which claims the benefit under 35 U.S.C. § 119(e) toProvisional Application Nos. 60/107,688, filed Nov. 9, 1998, 60/127,410,filed Apr. 1, 1999 and 60/134,016, filed May 12, 1999 and claimspriority under 35 U.S.C. § 119(a) to Great Britain Application No.9815879.3, Filing Date: Jul. 21, 1998, Great Britain Application No.9906712.6, filed Mar. 23, 1999 and Great Britain Application No.9909494.8, filed Apr. 23, 1999, which are incorporated herein byreference in their entireties.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a polynucleotide comprising aubiquitous chromatin opening element (UCOE) which is not derived from anLCR. The present invention also relates to a vector comprising thepolynucleotide sequence, a host cell comprising the vector, use of thepolynucleotide, vector or host cell in therapy and in an assay, and amethod of identifying UCOEs.

The current model of chromatin structure in higher eukaryotes postulatesthat genes are organised in “domains” (Dillon and Grosveld, 1994).Chromatin domains can consist of groups of genes that are expressed in astrictly tissue specific manner such as the human β-globin family(Grosveld et al., 1993), genes that are expressed ubiquitously such asthe human TBP/C5 locus (Trachtulec, Z. et al., 1997), or a mixture oftissue specific and ubiquitously expressed genes such as murine γ/βTCR/dad-1 locus, (Hong et al., 1997; Ortiz et al., 1997) and the humanα-globin locus, (Vyas et al., 1992). Genes with two different tissuespecificities may also be closely linked. For example, the human growthhormone and chorionic somatomammotropin genes (Jones et al., 1995).Chromatin domains are envisaged to exist in either a closed,“condensed”, transcriptionally silent state or in a “de-condensed”, openand transcriptionally competent configuration. The establishment of anopen chromatin structure characterised by DNase I sensitivity, DNAhypomethylation and histone hyperacetylation, is seen as a pre-requisiteto the commencement of gene expression.

The discovery of tissue-specific transcriptional regulatory elementsknown as locus control regions (LCRs) has provided novel insights intothe mechanisms by which a transcriptionally competent, open chromatindomain is established and maintained in certain cases. LCRs are definedby their ability to confer on a gene linked in cis host celltype-restricted, integration site independent, copy number-dependentexpression of the gene (Grosveld et al., 1987; Lang et al., 1988;Greaves et al., 1989; Diaz et al., 1994; Carson and Wiles, 1993; Boniferet al., 1990; Montoliu et al., 1996; Raguz et al., 1998; EP-A-0 332 667)especially as single copy transgenes (Ellis et al., 1996; Raguz et al.,1998). LCRs are able to obstruct the spread of heterochromatin andprevent position effect variegation (Festenstein et al., 1996; Milot etal., 1996). This pattern of expression conferred by LCRs suggests thatthese elements possess a powerful chromatin remodelling capability andare able to establish and maintain a transcriptionally competent, openchromatin domain. In addition, LCRs have been found to possess aninherent transcriptional activating capability that allows them toconfer tissue-specific gene expression independent of their cognatepromoter (Blom van Assendelft et al., 1989; Collis et al., 1990;Antoniou and Grosveld, 1990; Greaves et al., 1989).

All LCRs are associated with gene domains with a prominenttissue-specific or tissue restricted component and are associated with aseries of DNase I hypersensitive sites which can be located either 5′(Grosveld et al., 1987; Carson and Wiles, 1993; Bonifer et al., 1994;Jones et al., 1995; Montoliu et al., 1996) or 3′ (Greaves et al., 1989)of genes which they regulate. In addition, LCR elements have recentlybeen found to exist between closely spaced genes (Hong et al., 1997;Ortiz et al., 1997). An LCR-like element has also been reported to havean intronic location within a gene (Aronow et al., 1995). In the fewcases that have been investigated, these elements correspond to largeclusters of tissue-specific and ubiquitous transcription factor bindingsites (Talbot et al., 1990; Philipsen et al., 1990; Pruzina et al.,1991; Lake et al., 1990; Jarman et al., 1991; Aronow et al., 1995).

The discovery of LCRs suggests that the regulatory elements that controltissue-specific gene expression from a given chromatin domain areorganised in a hierarchical fashion. The LCR would appear to act as amaster switch wherein its activation results in the establishment of anopen chromatin structure that has to precede any gene expression.Transcription at the physiologically required level can then be achievedthrough a direct chromatin interaction between the LCR and the localpromoter and enhancer elements of an individual gene via looping out ofthe intervening DNA (Hanscombe et al., 1991; Wijgerde et al., 1995;Dillon et al., 1997).

As indicated above, an essential feature of an LCR is its tissuespecificity. The tissue specificity of an LCR has been investigated byOrtiz et al., (1997), wherein a number of DNase I hypersensitive sitesof the T-cell receptor alpha (TCR α) LCR were deleted and an LCR derivedelement, which opens chromatin in a number of tissues identified. Talbotet al., (1994, NAR, 22, 756-766) describe an LCR-like element that isconsidered to allow expression of a linked gene in a number of tissues.However, reproducible expression of the linked gene is not obtained. Thelevels of expression are indicated as having a standard deviation ofbetween 74% from the average value on a per-gene-copy basis where thegene is expressed where transgene copy number is 3 or more. When thecopy number is 1 or 2, the gene expression levels are 10 times lower andhave a standard deviation of 49% from the average value on aper-gene-copy basis where the gene is expressed. The element disclosedby Talbot et al., does not give reproducible expression of a linkedgene. This and the high variability of the system clearly limits the useof this system.

The long-term correction of genetically inherited disorders by genetherapy requires the maintenance and sustained expression of thetranscription unit at sufficiently high levels to be of therapeuticvalue. This, may be achieved by one of two approaches. Firstly,transcription units can be stably integrated into the host cell genomeusing, for example, retroviral (Miller, 1992; Miller et al., 1993) oradeno-associated viral (AAV) vectors (Muzyczka, 1992; Kotin, 1994;Flotte and Carter, 1995). Alternatively, therapeutic genes can beincorporated within self-replicating episomal vectors comprising viralorigins of replication such as those from EBV (Yates et al., 1985),human papovavirus BK (De Benedetti and Rhoads, 1991; Cooper and Miron,1993) and BPV-1 (Piirsoo et al., 1996).

Unfortunately, the level of expression that is normally seen from genesthat are integrated into the genome is too low or short in duration tobe of therapeutic value in most cases. This is due to what are generallyknown as “position effects”. The transcription of the introduced gene isdependent upon its site of integration where it comes under theinfluence of either competing activating (promoters/enhancers) or morefrequently, repressing (chromatin silencing) elements. Position effectscontinue to impose substantial constraints on the therapeutic efficacyof integrating virus-based vectors of retroviral and adeno-associatedviral (AAV) origin. Viral transcriptional regulatory elements arenotoriously susceptible to silencing by chromatin elements in thevicinity of integration sites. The inclusion of classical promoter andenhancer elements from highly expressed genes as part of the viralconstructs has not solved this major problem (Dai et al., 1992; Lee etal., 1993).

The inclusion of a fully functional LCR as part of the transcriptionunit overcomes this deficiency since this element can be used to drive apredictable, physiological and sustained level of expression of thedesired gene in a specific cell type (see Yeoman and Mellor, 1992;Brines and Klaus, 1993; Needham et al. 1992 and 1993; Tewari et al.,1998; Zhumabekov et al., 1995). This degree of predictability ofexpression is vital for a safe and successful gene therapy strategy.

The use of replicating episomal vectors (REVs) offers an attractivealternative to integrating viral vectors for producing long-term geneexpression. Firstly, REVs do not pose the same size limitations on thetherapeutic transcription unit as do viral vectors, with inserts inexcess of 300 kb being a possibility (Sun et al., 1994). Secondly, beingepisomal, REVs do not suffer from potential hazards associated withinsertional mutagenesis that is an inherent problem with integratingviral vectors. Lastly, REVs are introduced into the target cells usingnon-viral delivery systems that can be produced more cheaply at scalethan with viral vectors.

It has been demonstrated that both non-replicating, transientlytransfected plasmids (Reeves et al., 1985; Archer et al., 1992) and REVs(Reeves et al., 1985; Smith et al., 1993) assemble nucleosomes. Assemblyon REVs is more organised and resembles native chromatin whereasnucleosomes on transient plasmids are less well ordered and may allowsome access of transcription factors to target sequences although geneexpression can be inhibited (Archer et al., 1992). It has recently beendemonstrated that LCRs are able to confer long-term, tissue-specificgene expression from within REVs (International Patent Application WO98/07876). The generation of cultured mammalian cell lines producinghigh levels of a therapeutic protein product is a major developingindustry. Chromatin position effects make this a difficult, timeconsuming and expensive process. The most commonly used approach to theproduction of such mammalian “cell factories” relies on geneamplification induced by a combination of a drug resistance gene (e.g.DHFR, glutamine synthetase (Kaufman, 1990)) and high toxic drugconcentrations which have to be maintained at all times. The use ofvectors possessing LCRs from highly expressed gene domains, greatlysimplifies the generation of these cell lines (Needham et al., 1992;Needham et al., 1995).

A problem with the use of LCRs is that they are tissue specific andreproducible expression is only obtained in the specific cell type.Accordingly, one could not obtain reproducible expression in a tissuetype or a number of tissue types for which there is no LCR. Accordingly,there is a need for a UCOE, which is not derived from an LCR.

As indicated above, Ortiz et al., (1997) discloses an LCR derivedelement, which opens chromatin in number of tissues. There are a numberof problems with the LCR derived element of Ortiz et al., (1997). Inparticular, the element has to be carefully constructed usingrecombinant DNA techniques to contain the necessary regions of the LCRand also the element does not give reproducible levels of expression ofa linked gene in cells of different tissues types, especially when theelement is at single or low (less than 3) transgene copy number.

Elements comprising bi-directional promoters and methylation-free CpGislands have been disclosed; however, there is no disclosure orindication that the elements opens chromatin or maintain chromatin in anopen state and facilitate reproducible expression of an operably-linkedgene in cells of at least two different tissue types.

The human Surfeit locus spans approximately 60 kb and is located on9q34.2. The locus comprises bi-directional promoters between the SURF5and SURF3 genes and between the SURF1 and SURF2 genes (Huxley et al.,Mol. Cell. Biol., 10, 605-614, 1990; Duhig et al., Genomics, 52, 72-78,1998; Williams et al., Mol. Cell. Biol., 6, 4558-4569, 1986). There isno indication that these regions open chromatin or maintain chromatin inan open state and facilitate reproducible expression of anoperably-linked gene in cells of at least two different tissue types.

A bi-directional promoter is also disclosed by Brayton et al., (J. Biol.Chem., 269, 5313-5321, 1994) between the avian GPAT and AIRC genes.Again there is no indication that the region opens chromatin or maintainchromatin in an open state and facilitate reproducible expression of anoperably-linked gene in cells of at least two different tissue types.

A bi-directional promoter is disclosed by Ryan et al. (Gene, 196, 9-17,1997) between the mitochondrial chaperonin 60 and chaperonin 10 genes.Again there is no indication that the region opens chromatin or maintainchromatin in an open state and facilitate reproducible expression of anoperably-linked gene in cells of at least two different tissue types.

A bi-directional promoter is also disclosed associated with the murineHTF9 gene. Again there is no indication that the region opens chromatinor maintain chromatin in an open state and facilitate reproducibleexpression of an operably-linked gene in cells of at least two differenttissue types.

Palmiter et al., (PNAS USA, 95, 8428-8430, 1998) and InternationalPatent Application WO 94/13273 disclose an element associated with themetallothionein genes. The element comprises DNase I hypersensitivesites which are not associated with promoters. Furthermore, there is noevidence demonstrating that the element opens chromatin or maintainchromatin in an open state and facilitate reproducible expression of anoperably-linked gene in cells of at least two different tissue types.

The use of non-replicating, transiently transfected plasmids to achievegene expression by transfecting cells is well known. It is also knownthat only short term expression (generally less than 72 hours) isachieved using non-replicating, transiently transfected plasmids. Theshort term of expression is generally considered to be due to thebreakdown of the plasmid or loss of the plasmid from the cell. In viewof this drawback the use of such plasmids is limited.

The present invention provides isolated polynucleotides comprising aUCOE which opens chromatin or maintains chromatin in an open state andfacilitates reproducible expression of an operably-linked gene in cellsof at least two different tissue types, wherein the polynucleotide isnot derived from a locus control region. The isolated polynucleotidesaccording to the invention are preferably greater than about 1.5 kb inlength, more preferably greater than about 4 kb in length, when composedof endogenous genomic UCOE sequences. Functional composites of UCOEsequences, however, can be constructed from the endogenous genomic UCOE.Such composites can be less than 1.5 kb in length and are within thescope of the present invention.

A “locus control region” (LCR) is defined as a genetic element which isobtained from a tissue-specific locus of a eukaryotic host cell andwhich, when linked to a gene of interest and integrated into achromosome of a host cell, confers tissue-specific, integrationsite-independent, copy number-dependent expression on the gene ofinterest. A polynucleotide derived from an LCR can be any part or partsof an LCR. Preferably, a polynucleotide derived from an LCR is any partof an LCR that functions to open chromatin. An LCR is associated withone or more DNase I hypersensitive (HS) sites that are not associatedwith a promoter and it is preferred that the UCOE does not comprise HSsites that are not associated with a promoter. HS sites are well knownto those skilled in the art and can be identified based on the standardtechniques, which are described herein.

The term “facilitates reproducible expression” refers to the capabilityof the UCOE to facilitate reproducible activation of transcription ofthe operably-linked gene. The process is believed to involve the abilityof the UCOE to render the region of the chromatin encompassing the gene(or at least the transcription factor binding sites) accessible totranscription factors. Reproducible expression preferably means that thepolynucleotide when operably-linked to an expressible gene givessubstantially the same level of expression of the operably-linked geneirrespective of its chromatin environment and preferably irrespective ofthe cell tissue type. Preferably, substantially the same level ofexpression means a level of expression which has a standard deviationfrom an average value of less than 48%, more preferably less than 40%and most preferably, less than 25% on a per-gene-copy basis.Alternatively, substantially the same level of expression preferablymeans that the level of expression varies by less than 10 fold, morepreferably less than 5 fold and most preferably less than 3 fold on aper gene copy basis. The level of expression is preferably the level ofexpression measured in a transgenic animal. It is especially preferredthat the UCOE facilitates reproducible expression of an operably-linkedgene when present at a single or low (less than 3) copy number.

As used herein, “linked” refers to a cis-linkage in which the gene andthe UCOE are present in a cis relationship on the same nucleic acidmolecule. The term “operatively linked” refers to a cis-linkage in whichthe gene is subject to expression facilitated by the UCOE.

Open chromatin or chromatin in an open state refers to chromatin in ade-condensed state and is also referred to as euchromatin. Condensedchromatin is also referred to as heterochromatin. As indicated above,chromatin in a closed (condensed) state is transcriptionally silent.Chromatin in an open (de-condensed) state is transcriptionallycompetent. The establishment of an open chromatin structure ischaracterised by DNase I sensitivity, DNA hypomethylation and histonehyperacetylation. Standard methods for identifying open chromatin arewell known to those skilled in the art and are described in Wu, 1989,Meth. Enzymol., 170, 269-289; Crane-Robinson et al., 1997, Methods, 12,48-56; Rein et al., 1998, N.A.R., 26, 2255-2264.

The term “cells of two or more tissue types” refers to cells of at leasttwo, preferably at least 4 and more preferably all of the followingdifferent tissue types: heart, kidney, lung, liver, gut, skeletalmuscle, gonads, spleen, brain and thymus tissue. Preferably, thepolynucleotide facilitates reproducible expression non-tissuespecifically, i.e. with no tissue specificity. It is further preferredthat the polynucleotide of the present invention facilitatesreproducible expression in at least 50% and more preferably in alltissue types where active gene expression occurs.

Preferably, the polynucleotide of the present invention facilitatesreproducible expression of an operably-linked gene at a physiologicallevel. By physiological level, it is meant a level of gene expression atwhich expression in a cell, population of cells or a patient exhibits aphysiological effect. Preferably, the physiological level is an optimalphysiological level depending on the desired result. Preferably, thephysiological level is equivalent to the level of expression of anequivalent endogenous gene.

The UCOE of the present invention can be any element, which openschromatin or maintains chromatin in an open state and facilitatesreproducible expression of an operably-linked gene in cells of at leasttwo different tissue types provided it is not derived from an LCR. In apreferred embodiment, the UCOE comprises an extended methylation-free,CpG-island. CpG-islands have an average GC content of approximately 60%,compared with a 40% average in bulk DNA. One skilled in the art caneasily identify CpG-islands using standard techniques such as usingrestriction enzymes specific for C and G sequences. Such techniques aredescribed in Larsen et al., 1992 and Kolsto et al., 1986. An extendedmethylation-free CpG island is a methylation-free CpG island thatextends across a region encompassing more than one transcriptional startsite and/or extends for more than 300 bp and preferably more than 500bp.

Preferably, the UCOE is derived from a sequence that in its naturalendogenous position is associated with, more preferably, locatedadjacent to, a ubiquitously expressed gene. It is further preferred thatthe UCOE comprises at least one transcription factor binding site.Transcription factor binding sites include promoter sequences andenhancer sequences. Preferably, the UCOE comprises dual orbi-directional promoters that are divergently transcribed. Dualpromoters are defined herein as two or more promoters which areindependent from each other so that one of the promoters can beactivated or deactivated without effecting the other promoter orpromoters. A bi-directional promoter is defined herein as a region thatcan act as a promoter in both directions but cannot be activated ordeactivated in one direction only. Preferably, the UCOE comprises dualpromoters. Preferably, the UCOE comprises dual or bi-directionalpromoters that transcribe divergently (i.e. can lead to transcription inopposite directions) and which in their natural endogenous positions areassociated with ubiquitously expressed genes. Preferably, the UCOEcomprises dual promoters that are transcribe divergently. The UCOE maycomprise a heterologous promoter, i.e. a promoter that is not naturallyassociated with the other sequences of the UCOE. For example, it ispossible to use the CMV promoter with the UCOE associated with the hnRNPA2 and the HP1H-γ promoters, which is discussed further below. Thepresent invention therefore also provides a UCOE comprising one or moreheterologous promoters. The heterologous promoter or promoters canreplace of one or more of the endogenous promoters of the UCOE or can beused in addition to the one or more endogenous promoters of the UCOE.The heterologous promoter may be any promoter including tissue specificpromoters such as tumour-specific promoters and ubiquitous promoters.Preferably the heterologous promoter is a substantially ubiquitouspromoter and most preferably is the CMV promoter.

Preferably, the UCOE is not the 3725 bp Eco RI fragments comprising thebi-directional promoter of the Hpa II tiny fragment (HTF) island HTF9 asdescribed in Lavia et al., EMBO J., 6, 2773-2779, (1987).

Preferably, the UCOE is not the 149 bp MES-1 element located within a800 bp Bam HI genomic fragment located between the murine SURF1 andSURF2 genes of the Surfeit locus (Williams et al., Mol. Cell. Biol, 13,4784-4792, 1993). Preferably, the UCOE is not the bi-directionalpromoter located between the SURF5 and the SURF3 genes of the Surfeitlocus (Williams et al., Mol. Cell. Biol, 13, 4784-4792, 1993). It isfurther preferred that the UCOE is not derived from the human surfeitgene locus which spans 60 kb and is located on chromosome 9q34.2 asdefined in Duhig et al., Genomics, 52, 72-78, (1998) or thecorresponding murine locus (Huxley et al., Mol. Cell. Biol., 10,605-614, 1990).

Preferably, the UCOE is not the bi-directional promoter region locatedbetween avian GPAT and AIRC genes contained in the 1350 bp Sma Ifragment deposited in the GenBank database (accession no. L12533)(Gavalas et al., Mol. Cell. Biol., 13, 4784-4792, 1993) or thecorresponding human equivalent (Brayton et al., J. Biol. Chem., 269,5313-5321, 1994).

Preferably, the UCOE is not the 13894 bp genomic DNA fragment (GenBankaccession no. U68562) comprising the rat mitochondrial chaperonin 60 andchaperonin 10 genes. It is also preferred that the UCOE is not the 581bp fragment containing the bi-directional promoter located in theintergenic region between the rat mitochondrial chaperonin 60 andchaperonin 10 genes (Ryan et al., Gene, 196, 9-17, 1997).

In a preferred embodiment of the present invention, the UCOE is a 44 kbDNA fragment spanning the human TATA binding protein (TBP) gene and 12kb each of the 5′ and 3′ flanking sequence, or a functional homologue orfragment thereof.

A further preferred embodiment of the present invention, the UCOE is a60 kb DNA fragment spanning the human hnRNP A2 gene with 30 kb 5′flanking sequence and 20 kb 3′ flanking sequence, or a functionalhomologue or fragment thereof. In a further preferred embodiment, theUCOE comprises the sequence of FIG. 21 between nucleotides 1 to 6264 ora functional homologue or fragment thereof. This sequence encompassesthe hnRNP A2 promoter (nucleotides 5636 to 6264) and 5.5 kb 5′ flankingsequence comprising the HP1H-γ promoter.

In a further preferred embodiment of the present invention, the UCOE isa 25 kb DNA fragment spanning the human TBP gene with 1 kb 5′ and 5 kb3′ flanking sequence, or a functional homologue or fragment thereof.

In a further preferred embodiment, the UCOE is a 16 kb DNA fragmentspanning the human hnRNP A2 gene with 5 kb 5′ and 1.5 kb 3′ flankingsequence, or a functional homologue or fragment thereof.

In a further preferred embodiment, the UCOE comprises the sequence ofFIG. 21 between nucleotides 1 and 5636 (the 5.5 kb 5′ flanking sequenceof the hnRNP A2 promoter) and the CMV promoter or a functional homologueor fragment thereof.

In a further preferred embodiment, the UCOE comprises the sequence ofFIG. 21 between nucleotides 1 and 9127 or a functional homologue orfragment thereof. This sequence encompasses both the hnRNP A2 and HP1H-γpromoters and the 3′ flanking sequence of the hnRNP A2 promoter up tobut not including exon 2 of the hnRNP A2 gene.

It is further preferred that the UCOE of the present invention has thenucleotide sequence of FIG. 20 or FIG. 21, or a functional fragment orhomologue thereof.

The term “functional homologues or fragments” as used herein meanshomologues or fragments, which open chromatin or maintain chromatin inan open state and facilitate reproducible expression of anoperably-linked gene. Preferably, the homologues are species homologuescorresponding to the identified UCOEs or are homologues associated withother ubiquitously expressed genes. Sequence comparisons can be madebetween UCOEs in order to identify conserved sequence motifs enablingthe identification or synthesis of other UCOEs. Suitable softwarepackages for performing such sequence comparisons are well known tothose skilled in the art. A preferred software package for performingsequence comparisons is PCGENE (Intelligenetics, Inc. USA). Functionalfragments can be easily identified by methodically generating fragmentsof known UCOEs and testing for function. The identification of conservedsequence motifs will also assist in the identification of functionalfragments, as fragments comprising the conserved sequence motifs will belikely to be functional. Functional homologues also encompass modifiedUCOEs wherein elements of the UCOE have been replaced by similarelements, such as replacing one or more promoters of a UCOE withdifferent heterologous promoters. As indicated above, the heterologouspromoter may be any promoter including tissue specific promoters such astumour-specific promoters and ubiquitous promoters. Preferably theheterologous promoter is a strong and/or substantially ubiquitouspromoter and most preferably is the CMV promoter.

In another embodiment of the present invention, there is provided amethod for identifying a UCOE which facilitates reproducible expressionof an operably-linked gene in cells of at least two different tissuetypes, comprising: 1 .testing a candidate UCOE by transfecting cells ofat least two different tissue types with a vector containing thecandidate UCOE operably-linked to a marker gene; and 2.determining ifreproducible expression of the marker gene is obtained in the cells oftwo or more different tissue types.

Preferably, the method for identifying a UCOE of the present inventioncomprises the additional step of selecting candidate UCOEs that areassociated with one or more of: a ubiquitously expressed gene, a dual orbi-directional promoter and an extended methylation-free CpG-island.

Preferably, reproducible expression of the marker gene is determined incells containing a single copy of the UCOE linked to the marker gene.

The present invention further provides the method of the presentinvention wherein the candidate UCOE is tested by generating a non-humantransgenic animal containing cells comprising a vector containing thecandidate UCOE operably-linked to a marker gene and determining ifreproducible expression of the marker gene is obtained in the cells oftwo or more different tissue types. Preferably, the non-human transgenicanimal is a F1, or greater, generation non-human transgenic animal.Preferably the non-human transgenic animal is a rodent, more preferablya mouse.

The present invention provides a UCOE derivable from a nucleic acidsequence associated with or adjacent to a ubiquitously expressed gene.Preferably, the nucleic acid sequence comprises an extendedmethylation-free, CpG-island. It is further preferred that the nucleicacid sequence comprises at least one transcription factor binding site.Preferably, the nucleic acid sequence comprises dual or bi-directionalpromoters that are divergently transcribed. Preferably, the nucleic acidsequence comprises dual promoters that are divergently transcribed.Preferably, the nucleic acid sequence comprises dual or bi-directionalpromoters that are divergently transcribed and which are associated withubiquitously expressed genes. Preferably, the nucleic acid sequencecomprises dual promoters that are divergently transcribed and which areassociated with ubiquitously expressed genes.

The present invention also provides the use of the polynucleotide of thepresent invention, or a fragment thereof, in an assay for identifyingother UCOEs. Preferably, a fragment of the polynucleotide is used whichencompasses a conserved sequence or structural motif. Methods forperforming such an assay are well known to those skilled in the art.

The present invention provides a vector comprising the polynucleotide ofthe present invention. The vector preferably comprises an expressiblegene operably-linked to the polynucleotide. The expressible genecomprises the necessary elements enabling gene expression such assuitable promoters, enhancers, splice acceptor sequences, internalribosome entry site sequences (IRES) and transcription stop sites.Suitable elements for enabling gene expression are well known to thoseskilled in the art. The suitable elements for enabling gene expressioncan be the natural endogenous elements associated with the gene or maybe heterologous elements used in order to obtain a different level ortissue distribution of gene expression compared to the endogenous gene.Preferably, the vector comprises a promoter operably associated with theexpressible gene and the polynucleotide. The promoter may be a naturalendogenous promoter of the expressible gene or may be a heterologouspromoter. The heterologous promoter may be any promoter including tissuespecific promoters such as tumour-specific promoters and ubiquitouspromoters. Preferably the heterologous promoter is a strong and/or asubstantially ubiquitous promoter and most preferably is the CMVpromoter.

The vector may be any vector capable of transferring DNA to a cell.Preferably, the vector is an integrating vector or an episomal vector.

Preferred integrating vectors include recombinant retroviral vectors. Arecombinant retroviral vector will include DNA of at least a portion ofa retroviral genome which portion is capable of infecting the targetcells. The term “infection” is used to mean the process by which a virustransfers genetic material to its host or target cell. Preferably, theretrovirus used in the construction of a vector of the invention is alsorendered replication-defective to remove the effect of viral replicationof the target cells. In such cases, the replication-defective viralgenome can be packaged by a helper virus in accordance with conventionaltechniques. Generally, any retrovirus meeting the above criteria ofinfectiousness and capability of functional gene transfer can beemployed in the practice of the invention.

Suitable retroviral vectors include but are not limited to pLJ, pZip,pWe and pEM, well known to those of skill in the art. Suitable packagingvirus lines for replication-defective retroviruses include, for example,ψ Crip, ψ Cre, ψ 2 and ψ Am.

Other vectors useful in the present invention include adenovirus,adeno-associated virus, SV40 virus, vaccinia virus, HSV and poxvirusvectors. A preferred vector is the adenovirus. Adenovirus vectorsare well known to those skilled in the art and have been used to delivergenes to numerous cell types, including airway epithelium, skeletalmuscle, liver, brain and skin (Hitt, M M, Addison C L and Graham, F L(1997) Human adenovirus vectors for gene transfer into mammalian cells.Advances in Pharmacology 40: 137B206; and Anderson W F (1998) Human genetherapy. Nature 392(6679 Suppl): 25B30).

A further preferred vector is the adeno-associated (AAV) vector. AAVvectors are well known to those skilled in the art and have been used tostably transduce human T-lymphocytes, fibroblasts, nasal polyp, skeletalmuscle, brain, erythroid and heamopoietic stem cells for gene therapyapplications (Philip et al., 1994, Mol. Cell. Biol., 14, 2411-2418;Russell et al., 1994, PNAS USA, 91, 8915-8919; Flotte et al., 1993, PNASUSA, 90, 10613-10617; Walsh et al., 1994, PNAS USA, 89, 7257-7261;Miller et al., 1994, PNAS USA, 91, 10183-10187; Emerson, 1996, Blood,87, 3082-3088). International Patent Application WO 91/18088 describesspecific AAV based vectors.

Preferred episomal vectors include transient non-replicating episomalvectors and self-replicating episomal vectors with functions derivedfrom viral origins of replication such as those from EBV, humanpapovavirus (BK) and BPV-1. Such integrating and episomal vectors arewell known to those skilled in the art and are fully described in thebody of literature well known to those skilled in the art. Inparticular, suitable episomal vectors are described in WO98/07876.

Mammalian artificial chromosomes are also preferred vectors for use inthe present invention. The use of mammalian artificial chromosomes isdiscussed by Calos (1996, TIG, 12, 463-466).

In a preferred embodiment, the vector of the present invention is aplasmid. It is further preferred that the plasmid is a non-replicating,non-integrating plasmid.

The term “plasmid” as used herein refers to any nucleic acid encoding anexpressible gene and includes linear or circular nucleic acids anddouble or single stranded nucleic acids. The nucleic acid can be DNA orRNA and may comprise modified nucleotides or ribonucleotides, and may bechemically modified by such means as methylation or the inclusion ofprotecting groups or cap- or tail structures.

A non-replicating, non-integrating plasmid is a nucleic acid which whentransfected into a host cell does not replicate and does notspecifically integrate into the host cell's genome (i.e. does notintegrate at high frequencies and does not integrate at specific sites).

Replicating plasmids can be identified using standard assays includingthe standard replication assay of Ustav et al., EMBO J., 10, 449-457,1991.

Preferably, a non-replicating, non-integrating plasmid is a plasmid thatcannot be stably maintained in cells, independently of genomic DNAreplication, and which does not persist in progeny cells for three ormore cell divisions without a significant loss in copy number of theplasmid in the cells, i.e., with a loss of greater than an average ofabout 50% of the plasmid molecules in progeny cells between a given celldivision. Generally, in self-replicating vectors, the self-replicatingfunction is provided by using a viral origin of replication andproviding one or more viral replication factors that are required forreplication mediated by that particular viral origin. Self-replicatingvectors are described in WO 98/07876. The term “transientlytransfecting, non-integrating plasmid” herein means the same as the term“non-replicating, non-integrating plasmid” as defined above.

Preferably the plasmid is a naked nucleic acid. As used herein, the term“naked” refers to a nucleic acid molecule that is free of directphysical associations with proteins, lipids, carbohydrates orproteoglycans, whether covalently or through hydrogen bonding. The termdoes not refer to the presence or absence of modified nucleotides orribonucleotides, or chemical modification of the all or a portion of anucleic acid molecule by such means as methylation or the inclusion ofprotecting groups or cap- or tail structures.

Preferably, the vector of the present invention comprises the sequenceof FIG. 21 between nucleotides 1 and 7627 (encompassing both the hnRNPA2 and HP1H-γ promoters), the CMV promoter, a multiple cloning site, apolyadenylation sequence and genes encoding selectable markers undersuitable control elements. Preferably the vector of the presentinvention is the CET200 or the CET210 vector schematically shown in FIG.49.

The present invention also provides a host cell transfected with thevector of the present invention. The host cell may be any cell such asyeast cells, insect cells, bacterial cells and mammalian cells.Preferably the host cell is a mammalian cell and may be derived frommammalian cell lines such as the CHO cell line, the 293 cell line andN50 cells.

Preferably, the operably-linked gene is a therapeutic nucleic acidsequence. Therapeutically useful nucleic acid sequences, which may beused in the present invention, include sequences encoding receptors,enzymes, ligands, regulatory factors, hormones, antibodies or antibodyfragments and structural proteins. Therapeutic nucleic acid sequencesalso include sequences encoding nuclear proteins, cytoplasmic proteins,mitochondrial proteins, secreted proteins, membrane-associated proteins,serum proteins, viral antigens, bacterial antigens, protozoal antigensand parasitic antigens. Nucleic acid sequences useful according to theinvention also include sequences encoding proteins, peptides,lipoproteins, glycoproteins, phosphoproteins and nucleic acid (e.g.,RNAs or antisense nucleic acids). Proteins or polypeptides which can beencoded by the therapeutic nucleic acid sequence include hormones,growth factors, enzymes, clotting factors, apolipoproteins, receptors,erythropoietin, therapeutic antibodies or fragments thereof, drugs,oncogenes, tumor antigens, tumor suppressors, viral antigens, parasiticantigens and bacterial antigens. Specific examples of these compoundsinclude proinsulin, growth hormone, androgen receptors, insulin-likegrowth factor I, insulin-like growth factor II, insulin-like growthfactor binding proteins, epidermal growth factor, transforming growthfactor-α, transforming growth factor-β, platelet-derived growth factor,angiogenesis factors (acidic fibroblast growth factor, basic fibroblastgrowth factor, vascular endothelial growth factor and angiogenin),matrix proteins (Type IV collagen, Type VII collagen, laminin),phenylalanine hydroxylase, tyrosine hydroxylase, oncoproteins (forexample, those encoded by ras, fos, myc, erb, src, neu, sis, jun), HPVE6 or E7 oncoproteins, p53 protein, Rb protein, cytokine receptors,IL-1, IL-6, IL-8, and proteins from viral, bacterial and parasiticorganisms which can be used to induce an immunological response, andother proteins of useful significance in the body. The choice of gene,to be incorporated, is only limited by the availability of the nucleicacid sequence encoding it. One skilled in the art will readily recognisethat as more proteins and polypeptides become identified they can beintegrated into the polynucleotide of the present invention andexpressed.

When the polynucleotide of the present invention is comprised in aplasmid, it is preferred that the plasmid be used in monogenic genetherapy such as in the treatment of Duchenne muscular dystrophy and inDNA vaccination and immunisation methods.

The polynucleotide of the invention also may be used to express genesthat are already expressed in a host cell (i.e., a native or homologousgene), for example, to increase the dosage of the gene product. Itshould be noted, however, that expression of a homologous gene mightresult in deregulated expression, which may not be subject to control bythe UCOE due to its over-expression in the cell.

The polynucleotide of the invention may be inserted into the genome of acell in a position operably associated with an endogenous (native) geneand thereby lead to increased expression of the endogenous gene. Methodsfor inserting elements into the genome at specific sites are well knownto those skilled in the art and are described in U.S. Pat. No. 5,578,461and U.S. Pat. No. 5,641,670. Alternatively, the polynucleotide of thepresent invention in its endogenous (native) position on the genome mayhave a gene inserted in an operably associated position so thatexpression of the gene occurs. Again, methods for inserting genes intothe genome at specific sites are well known to those skilled in the artand are described in U.S. Pat. No. 5,578,461 and U.S. Pat. No.5,641,670.

The present invention provides the use of the polynucleotide of thepresent invention to increase the expression of an endogenous genecomprising inserting the polynucleotide into the genome of a cell in aposition operably associated with the endogenous gene thereby increasingthe level of expression of the gene.

Numerous techniques are known and are useful according to the inventionfor delivering the vectors described herein to cells, including the useof nucleic acid condensing agents, electroporation, complexation withasbestos, polybrene, DEAE cellulose, Dextran, liposomes, cationicliposomes, lipopolyamines, polyornithine, particle bombardment anddirect microinjection (reviewed by Kucherlapati and Skoultchi, Crit.Rev. Biochem. 16:349-379 (1984); Keown et al., Methods Enzymol. 185:527(1990)).

A vector of the invention may be delivered to a host cellnon-specifically or specifically (i.e., to a designated subset of hostcells) via a viral or non-viral means of delivery. Preferred deliverymethods of viral origin include viral particlepackaging cell lines astransfection recipients for the vector of the present invention intowhich viral packaging signals have been engineered, such as those ofadenovirus, herpes viruses and papovaviruses. Preferred non-viral basedgene delivery means and methods may also be used in the invention andinclude direct naked nucleic acid injection, nucleic acid condensingpeptides and non-peptides, cationic liposomes and encapsulation inliposomes.

The direct delivery of vector into tissue has been described and someshort term gene expression has been achieved. Direct delivery of vectorinto muscle (Wolff et al., Science, 247, 1465-1468, 1990) thyroid (Sykeset al., Human Gene Ther., 5, 837-844, 1994) melanoma (Vile et al.,Cancer Res., 53, 962-967, 1993), skin (Hengge et al., Nature Genet, 10,161-166, 1995), liver (Hickman et al., Human Gene Therapy, 5, 1477-1483,1994) and after exposure of airway epithelium (Meyer et al., GeneTherapy, 2, 450-460, 1995) is clearly described in the prior art.

Various peptides derived from the amino acid sequences of viral envelopeproteins have been used in gene transfer when co-administered withpolylysine DNA complexes (Plank et al., J. Biol. Chem. 269:12918-12924(1994));. Trubetskoy et al., Bioconjugate Chem. 3:323(1992); WO91/17773; WO 92/19287; and Mack et al., Am. J. Med. Sci. 307:138-143(1994)) suggest that co-condensation of polylysine conjugates withcationic lipids can lead to improvement in gene transfer efficiency.International Patent Application WO 95/02698 discloses the use of viralcomponents to attempt to increase the efficiency of cationic lipid genetransfer.

Nucleic acid condensing agents useful in the invention include spermine,spermine derivatives, histones, cationic peptides, cationic non-peptidessuch as polyethyleneimine (PEI) and polylysine. Spermine derivativesrefers to analogues and derivatives of spermine and include compounds asset forth in International Patent Application. WO 93/18759 (publishedSep. 30, 1993).

Disulphide bonds have been used to link the peptidic components of adelivery vehicle (Cotten et al., Meth. Enzymol. 217:618-644 (1992)); seealso, Trubetskoy et al. (supra).

Delivery vehicles for delivery of DNA constructs to cells are known inthe art and include DNA/poly-cation complexes which are specific for acell surface receptor, as described in, for example, Wu and Wu, J. Biol.Chem. 263:14621 (1988); Wilson et al., J. Biol. Chem. 267:963(1992); andU.S. Pat. No. 5,166,320).

Delivery of a vector according to the invention is contemplated usingnucleic acid condensing peptides. Nucleic acid condensing peptides,which are particularly useful for condensing the vector and deliveringthe vector to a cell, are described in WO 96/41606. Functional groupsmay be bound to peptides useful for delivery of a vector according tothe invention, as described in WO 96/41606. These functional groups mayinclude a ligand that targets a specific cell-type such as a monoclonalantibody, insulin, transferrin, asialoglycoprotein, or a sugar. Theligand thus may target cells in a non-specific manner or in a specificmanner that is restricted with respect to cell type.

The functional groups also may comprise a lipid, such as palmitoyl,oleyl, or stearoyl; a neutral hydrophilic polymer such as polyethyleneglycol (PEG), or polyvinylpyrrolidine (PVP); a fusogenic peptide such asthe HA peptide of influenza virus; or a recombinase or an integrase. Thefunctional group also may comprise an intracellular trafficking proteinsuch as a nuclear localisation sequence (NLS) and endosome escape signalor a signal directing a protein directly to the cytoplasm.

The present invention also provides the polynucleotide, vector or hostcell of the present invention for use in therapy.

Preferably, the polynucleotide, vector or host cell is used in genetherapy.

The present invention also provides the use of the polynucleotide,vector or host cell of the present invention in the manufacture of acomposition for use in gene therapy.

The present invention also provides a method of treatment, comprisingadministering to a patient in need of such treatment an effective doseof the polynucleotide, vector or host cell of the present invention.Preferably, the patient is suffering from a disease treatable by genetherapy.

The present invention also provides a pharmaceutical compositioncomprising the polynucleotide, vector or host cell of the presentinvention in combination with a pharmaceutically acceptable recipient.

The present invention also provides use of a polynucleotide, vector orhost cell of the present invention in a cell culture system in order toobtain the desired gene product. Suitable cell culture systems are wellknown to those skilled in the art and are fully described in the body ofliterature known to those skilled in the art.

The present invention also provides the use of the polynucleotide of thepresent invention in producing transgenic plant genetics. The generationof transgenic plants which have increased yield, resistance, etc. arewell known to those skilled in the art. The present invention alsoprovides a transgenic plant containing cells which contain thepolynucleotide of the present invention.

The present invention also provides a transgenic non-human animalcontaining cells, which contain the polynucleotide of the presentinvention.

The pharmaceutical compositions of the present invention may comprisethe polynucleotide, vector or host cell of the present invention, ifdesired, in admixture with a pharmaceutically acceptable carrier ordiluent, for therapy to treat a disease or provide the cells of aparticular tissue with an advantageous protein or function.

The polynucleotide, vector or host cell of the invention or thepharmaceutical composition may be administered via a route whichincludes systemic intramuscular, intravenous, aerosol, oral (solid orliquid form), topical, ocular, as a suppository, intraperitoneal and/orintrathecal and local direct injection.

The exact dosage regime will, of course, need to be determined byindividual clinicians for individual patients and this, in turn, will becontrolled by the exact nature of the protein expressed by the gene ofinterest and the type of tissue that is being targeted for treatment.

The dosage also will depend upon the disease indication and the route ofadministration. Advantageously, the duration of treatment will generallybe continuous or until the cells die. The number of doses will dependupon the disease, and efficacy data from clinical trials.

The amount of polynucleotide or vector DNA delivered for effective genetherapy according to the invention will preferably be in the range ofbetween about 50 ng-1000 μg of vector DNA/kg body weight; and morepreferably in the range of between about 1-100 μg vector DNA/kg.

Although it is preferred according to the invention to administer thepolynucleotide, vector or host cell to a mammal for in vivo cell uptake,an ex vivo approach may be utilised whereby cells are removed from ananimal, transduced with the polynucleotide or vector, and thenre-implanted into the animal. The liver, for example, can be accessed byan ex vivo approach by removing hepatocytes from an animal, transducingthe hepatocytes in vitro and re-implanting the transduced hepatocytesinto the animal (e.g., as described for rabbits by Chowdhury et al.,Science 254:1802-1805, 1991, or in humans by Wilson, Hum. Gene Ther.3:179-222, 1992). Such methods also may be effective for delivery tovarious populations of cells in the circulatory or lymphatic systems,such as erythrocytes, T cells, B cells and haematopoietic stem cells.

In another embodiment of the invention, there is provided a mammalianmodel for determining the tissue-specificity and/or efficacy of genetherapy using the polynucleotide, vector or host cell of the invention.The mammalian model comprises a transgenic animal whose cells containthe vector of the present invention. Methods of making transgenic mice(Gordon et al., Proc. Natl. Acad. Sci. USA 77:7380 (1980); Harbers etal., Nature 293:540 (1981); Wagner et al., Proc. Natl. Acad. Sci. USA78:5016 (1981); and Wagner et al., Proc. Natl. Acad. Sci. USA 78:6376(1981), sheep, pigs, chickens (see Hammer et al., Nature 315:680(1985)), etc., are well-known in the art and are contemplated for useaccording to the invention. Such animals permit testing prior toclinical trials in humans.

Transgenic animals containing the polynucleotide of the invention alsomay be used for long-term production of a protein of interest.

The present invention also relates to the use of the polynucleotide ofthe present invention in functional genomics applications. Functionalgenomics relates principally to the sequencing of genes specificallyexpressed in particular cell types or disease states and now providesthousands of novel gene sequences of potential interest for drugdiscovery or gene therapy purposes. The major problem in using thisinformation for the development of novel therapies lies in how todetermine the functions of these genes. UCOEs can be used in a number offunctional genomic applications in order to determine the function ofgene sequences. The functional genomic applications of the presentinvention include, but are not limted to:

(1)Using the polynucleotide of the present invention to achievesustained expression of anti-sense versions of the gene sequences orribozyme knockdown libaries, thereby determining the effects ofinactivating the gene on cell phenotype.

(2)Using the polynucleotide of the present invention to prepareexpression libraries for the gene sequences, such that delivery intocells will result in reliable, reproducible, sustained expression of thegene sequences. The resulting cells, expressing the gene sequences canbe used in a variety of approaches to function determination and drugdiscovery. For example, raising antibodies to the gene product forneutralisation of its activity; rapid purification of the proteinproduct of the gene itself for use in structural, functional or drugscreening studies; or in cell-based drug screening.

(3)Using the polynucleotide of the present invention in approachesinvolving mouse embryonic stem (ES) cells and transgenic mice. One ofthe most powerful functional genomics approaches involves randominsertion into genes in mouse ES cells of constructs which only allowdrug selection following insertion into expressed genes, and which canreadily be rescued for sequencing (G. Hicks et al., 1997, NatureGenetics, 16, 338-344). Transgenic mice with knockout mutations in geneswith novel sequences can then readily be made to probe their function.At present this technology works well for the 10% of mouse genes whichare well expressed in mouse ES cells. Incorporation of UCOEs into theintegrating constructs will enable this technique to be extended toidentify all genes expressed in mice.

The following examples, with reference to the figures, are offered byway of illustration and are not intended to limit the invention in anymanner. The preparation, testing and analysis of several representativepolynucleotides of the invention are described in detail below. One ofskill in the art may adapt these procedures for preparation and testingof other polynucleotides of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the human TBP gene locus.

A: Schematic representation of the pCYPAC-2 clones containing the humanTBP gene used in this study. The positions of Not I and Sac lIrestriction sites that may indicate the positions of unidentified genesare marked.

B: Illustration of the CpG-island spanning the 5=TBP/C5 regions. Thedensity of CpG di-nucleotide residues implies that the methylation-freeisland is 3.4 kb in length and extends between the FspI site withinintron I of C5, and the HindIII site within the first intron of TBP.

C: Is a further schematic representation of the clones from the TBP/C5region. The arrangement of the genes has been reversed from that givenin FIG. 1A. Please note, the C5 gene is also referred to as the PSMB1gene. A 257 kb contiguous region from the telomere of chromosome 6q withpositions of the 3 closely linked genes and relevant restriction sitesis shown (B, Bss HII; N, NotI; S, SaclI). PAC clones with theirdesignated names are indicated. The subclone pBL3-TPO-puro is alsoshown. The distance between the NotI site within the first exon of PDCD2and the beginning of the telomeric repeat is approximately 150 kb.

FIG. 2 shows end-fragment analysis of TLN:3 and TLN:8 transgenic mice.Southern blot analysis of transgenic mouse tail biopsy DNA samples wereprobed with small DNA fragments located at (a) the 3′ end of thetransgene, (b) the 5′ end, (c) the promoter, (d) −7.7 kb from TBP mRNACAP site, (e) −12 kb from TBP mRNA CAP site. The results for TLN:3 (a,b)show that there is only one hybridising band with both end-probes, whichdoes not match the predicted size for any head-to-head, head-to-tail, ortail-to-tail concatamer. Thus it would appear that there is only onetransgene copy in this line. However, panel (c) shows that with apromoter probe, two bands are seen indicating that there must also be asecond, deleted copy of the transgene present in this line. TLN:8analysis in (a) shows a transgene concatamer band at 6 kb and an endfragment band at 7.8 kb. As the concatamer band is twice the intensityof the end fragment, this indicates a copy number of three for thisline. The lack of hybridisation in (b) suggests a deletion at the 5′ endof all three copies has occurred and work is in progress to map this.Panels (d) and (e) indicate that the transgenes appear to be intact upto 12 kb 5′ to the TBP gene.

FIG. 3A shows the analysis of TLN:28 mice. Southern blots of TLN:28 DNAwere hybridised to a probe located at the very 3′ end of the transgenelocus. Multiple bands were seen to hybridise to this probe, suggestingmultiple integration events. However, an intense concatamer band is seenin the position expected for a head to tail integration event.Comparison of the signal intensities between this and the end-fragmentssuggested a copy number of approximately 4 in this line.

FIG. 3B shows a summary of transgene organisation in TLN mouse lines.TLN:3: contains two copies of the transgene in a head to tailarrangement. A deletion has occurred at both the 5′ and 3′ ends of thisarray. The 5′ deletion extends into the 5′ flanking region of TBP,completely deleting the C5 gene in this copy. At the 3′ end, thedeletion extends into the 3′UTR of TBP, leaving the C5 gene intact. Thisanimal, therefore, possesses a single copy of the C5 gene and a singlefunctional copy of the TBP gene. TLN:8: contains a head to tailarrangement of three copies. Each copy would seem to possess a deletionat the very 5′ region, although the extent of this deletion is not knownat present, it does not extend to the C5 gene as human C5 mRNA isdetected in this line. TLN:28: contain 5 copies in a head to tailconfiguration, but there are also a number of additional fragments seen,indicating that this array may be more complex.

FIG. 3C shows an updated summary of the transgene organisation in theTLN mouse lines. The figure shows the predicted organisations of the TLNtransgene arrays in each of the mouse lines. Only functional genes areshown and only one of the 3 possible arrangements of the TLN:3 mice isindicated.

FIG. 4 shows analysis of the deletion in TLN:3 mice. A series of probeswere hybridised to Southern blots of TLN:3 DNA. Only the furthest 5′probe gave a single band, indicating that the deleted copy did notcontain this sequence. The deletion maps to a region upstream of themajor TBP mRNA CAP sites, Ets factor binding site and DNase Ihypersensitive site. It is currently unknown if the entire 5′ region isdeleted in this copy or a small internal deletion has occurred.

FIG. 5 shows the comparison of TBP and C5 mRNA sequences from human andmouse. (a) The human C5 mRNA sequence (SEQ. ID NO:23) from nt. 358 to708 (Genbank accession no. D00761) exhibits significant homology to themouse sequence (SEQ. ID NO:24) (indicated by a vertical bar) from nt.355 to 705 (Genbank accession no. X80686). RT-PCR amplification of bothhuman and mouse mRNAs produces a mixture of 350 bp DNA molecules fromboth species. The primer locations (highlighted, 5′ primer C5RTF, 3′primer C5R) are positioned so as to span a number of exons, eliminatingerror from PCR amplification from contaminating genomic DNA. Althoughthe intron/exon structure of either the human or mouse gene is limited,the distance between the primers is such that they are positioned indifferent exons. Mouse and human PCR products can be distinguished byincubation with PstI that will only cut the mouse sequence.Radiolabelling of the C5RTF primer gives a product of 173nt whenresolved on a denaturing polyacrylamide gel. (b) Similar analysis forhuman TBP mRNA sequence (SEQ. ID NO:25) from nt. 901 in exon 5 to nt.1185 in exon 7 (Genbank accession no. M55654) and mouse TBP mRNA (SEQ.ID NO:26) from positions 655 to 939 (Genbank accession no. D01034). Thelast nucleotide from an exon and the first nucleotide from the next exonare shown in red. The primers used (highlighted) were 5′ TB-22 and 3′TB-14. The size of the amplified product from both species with theprimers shown (boxed) is 284 bp. The Bsp 14071 site 63 nt from the 5′end of the PCR products allows human and mouse transcripts to bedistinguished. The size of the human specific product on apolyacrylamide gel with radiolabelled TB-14 is 221nt.

FIG. 6 shows expression analysis of human TBP expression in the TLNtransgenic mice. Total RNA (1 μg) from various mouse tissues was used ina reverse transcription reaction using Avian Myeloblastosis Virusreverse transcriptase. As a control, human RNA from K562 cells andnon-transgenic mouse RNA were also used. (a)Location of the recognitionsite for the human specific restriction endonucleases within the TB22/14RT-PCR products. (b)Analysis of TLN:3 expression in various tissues. Ascan be seen, the level of human expression is physiological in alltissues. (c) Similar analysis for TLN:8. (d)Analysis of TLN:28 indicateslevels of human TBP mRNA are again expressed at comparable levels to theendogenous gene.

FIG. 7 shows expression analysis of human C5 expression in the TLNtransgenic mice. Analysis was performed as in FIG. 6. The upper panel(a) shows the location of the recognition site for the mouse specificrestriction endonucleases within the C5RTF/C5R RT-PCR products. (b)Analysis of C5 expression in various tissues of TLN transgenics can beseen, the level of human expression is physiological in all tissuestested.

FIG. 8 shows a summary of quantification of (a) human TBP geneexpression (b) human C5 gene expression in TLN transgenic mice.

FIG. 9 shows a schematic representation of the pWE-TSN cosmid.

FIG. 10 shows transgene copy number determination of pWE-TSN L-cellclones. Mouse L-cells were transfected with the pWE-TSN cosmid, DNAisolated and used to generate Southern blots. Blots were probed with aDNA fragment from the two copy murine vavlocus and a probe located −7 kbfrom the TBP gene. Copy numbers were determined from the ratio of thethree copy TLN:8 control and are given underneath each lane. Copynumbers ranged from 1 to 60.

FIG. 11 shows a summary of expression of pWE-TSN cosmid clones in mouseL-cells.

FIG. 12 shows DNase I hypersensitive site analysis of the human TBPlocus. Probes located over a 40 kb region surrounding the TBP gene wereused to probe Southern blots of K562 nuclei digested with increasingconcentrations of DNase I. Only two hypersensitive sites were found, atthe promoters of the PSMB1 and the TBP gene. Increased DNase Iconcentration is shown from left to right in all cases.

FIG. 13A shows a schematic representation of the human hnRNP A2 genelocus showing the large 160 kb pCYPAC-derived clone MA160. The reversearrow denotes the HP1H-γ gene. The two Sac lI sites, which may representthe presence of methylation-free islands are boxed.

FIG. 13B shows the 60 kb Aat II sub-fragment derived from MA160. Both ofthese have been used for generation of transgenic mice.

FIG. 13C shows the extent of the CpG-island (red bar) spanning the 5′end of the hnRNP A2 gene. The CpG residues are denoted as verticallines. The numbers are in relation to the transcriptional start site(+1) of the hnRNP A2 gene (solid arrow). The broken arrow denotes theposition of the divergently transcribed HP1H-γ gene. The 16 kbsub-fragment that contains the intact hnRNP A2 gene is also shown.

FIG. 14A shows exons 10 to 12 of the human hnRNP A2 cDNA (SEQ ID NO:27),and FIG. 14B shows quantification of human and mouse hnRNP A2 geneexpression. Human (K562) and mouse RNA was reverse transcribed with aprimer to exon 12 of the hnRNPA2 gene. Samples were subsequentlyamplified by PCR with primers Hn9 and Hn11 spanning exons 10 to 12. Theproduct produced was then digested with random enzymes to find a cutsite unique to each species. The mouse product can be seen to contain aHindIII that is not present in the human product.

FIG. 15 shows the analysis of human hnRNP A2 expression in transgenicmice microinjected with the Aa60 fragment (FIG. 13B). Total RNA fromvarious tissues was analysed as described in FIG. 15. After RT-PCR,samples were either untreated (−) or digested with HindIII (+) and thenseparated on a polyacrylamide gel to resolve the human (H) and mouse (M)products. Intensity of the bands was measured by PhosphorImageranalysis.

FIG. 16 shows the analysis of human hnRNP A2 expression by transgenicmice microinjected with the 160 kb Nru I fragment (FIG. 13A). Atransgenic mouse was dissected and total RNA extracted from tissues. TheRNA was reverse transcribed by Hn11 and then amplified by PCR usingprimers Hn9 and Hn11 of which Hn9 was radioactively end-labelled with³²P. Samples were either untreated (−) or digested with HindIII (+) andthen separated on a 5% polyacrylamide gel in the presence of 8M urea asdenaturant to resolve the human (H) and mouse (M) products. Intensity ofthe bands was measured by PhosphorImager analysis.

FIG. 17 shows the quantification of hnRNP A2 transgene expression. TheRT-PCR analysis of human hnRNP A2 transgene expression in various mousetissues was quantified by PhosphorImager. Levels are depicted as apercentage of murine hnRNP A2 expression on a transgene copy numberbasis. A: Mice harbouring MA160 (see FIG. 15). B: Mice harbouring Aa60(see FIG. 16).

FIG. 18 shows DNase I hypersensitive site mapping of the human hnRNP A2gene locus. Nuclei from K562 cells were digested with increasingconcentrations of DNase I. DNA from these nuclei was subsequentlydigested with a combination of Aat II and Nco I restrictionendonucleases and Southern blotted. The blot was then probed with a 766bp Eco RI/Nco I fragment from exon II of the hnRNP A2 gene. Threehypersensitive sites were identified corresponding to positions B1.1,−0.7 and B0.1 kb 5′ of the hnRNP A2 transcriptional start site.

FIG. 19 shows the bioinformatic analysis and sequence comparisonsbetween the hnRNP A2 and the TBP loci.

FIG. 20 shows the nucleotide sequence of a genomic clone of the TBPlocus (SEQ. ID NO:28) beginning at the 5′ HindIII site (nucleotides 1 to9098).

FIG. 21 shows the nucleotide sequence of a genomic clone of the hnRNP A2locus (SEQ. ID NO:29) beginning at the 5′ Hind III site shown in FIG. 22(nucleotides 1 to 15071).

FIG. 22 shows the expression vectors containing sub-fragments located inthe dual promoter region between RNP and HP1H-γ which were designedusing both GFP and a Neo^(R) reporter genes. The vectors are: a controlvector with the RNP promoter (RNP) driving GFP/Neo expression; a vectorcomprising the 5.5 kb fragment upstream of the RNP promoter region andthe RNP promoter (5.5RNP); vectors constructed using a splice acceptorstrategy wherein the splice acceptor/branch concensus sequences (derivedfrom exon 2 of the RNP gene) were cloned in front of the GFP gene,resulting in exon 1/part of intron 1 upstream of GFP (7.5RNP, carryingapproximately 7.5 kb of the RNP gene preceding the GFP gene; and avector comprising the 1.5 kb fragment upstream of the RNP promoterregion and the RNP promoter (1.5RNP).

FIG. 23 shows expression vectors containing sub-fragments located in thedual promoter region between RNP and HP1H-γ which were designed usingboth GFP and a Neo^(R) reporter genes. The vectors comprise theheterologous CMV promoter. The vectors are: control vectors with the CMVpromoter driving GFP/Neo expression with (a) internal ribosome entrysite sequences (CMV-EGFP-IRES) and (b) with without internal ribosomeentry site sequences and an SV40 promoter upstream of the Neo^(R)reporter gene (CMV-EGFP); a vector comprising the 5.5 kb fragmentupstream of the RNP promoter region and the CMV promoter driving GFP/Neoexpression with internal ribosome entry site sequences (5.5CMV); avector comprising 4.0 kb sequence encompassing the RNP and the HP1H-γpromoters and the CMV promoter driving GFP/Neo expression with an SV40promoter upstream of the Neo^(R) reporter gene (4.0CMV); and a vectorcomprising 7.5 kb sequences of the RNP gene including exon 1 and part ofintron 1, and the CMV promoter driving GFP-Neo expression with an SV40promoter upstream of the Neo^(R) reporter gene (7.5CMV).

FIG. 24 shows the number of G418^(R) colonies produced by transfectingthe RNP- and CMV-constructs into CHO cells.

FIG. 25 shows the comparison of GFP expression in G418-selected CHOclones transfected with RNP- and CMV-constructs with and withoutupstream elements.

FIG. 26 shows the average median GFP fluorescence levels inG418-selected CHO clones transfected with RNP-constructs with andwithout upstream elements over a period of 40 days.

FIG. 27 shows FACS profiles of GFP expression of CMV-GFP pools culturedin the absence of G418 followed over a period of 103 days.

FIG. 28 shows FACS profiles of GFP expression of 5.5CMV-GFP poolscultured in the absence of G418 followed over a period of 103 days.

FIG. 29 shows the percentage of transfected cells expressing GFPreducing over a 68 day time course.

FIG. 30 shows the median fluorescence of G418 selected cells transfectedwith CMV-constructs over a 66 day time course.

FIG. 31 shows the percentage of positive G418 selected cells transfectedwith CMV-constructs over a 66 day time course.

FIG. 32 shows the median fluorescence of G418 selected cells transfectedwith CMV-constructs on day 13 after transfection.

FIG. 33 shows the percentage of positive G418 selected cells transfectedwith CMV-constructs over a 27 day time course.

FIG. 34 shows the colony numbers after transfection of CHO cells withvarious CMV-constructs.

FIG. 35 shows the dot blot analysis of human PSMB1, PDCD2 and TBP mRNAs.The tissue distribution of mRNAs from genes within the TBP cluster usinga human multiple tissue mRNA dotbblot: each segment is loaded with agiven amount of poly(A)⁺ RNA (A, shown in ng below each tissue). Thedot-blot was hybridised with (B) PSMB1 cDNA, (C) a 4.7 kb genomicfragment (MA445) containing a partial PDCD2 gene and (D) TBP cDNA. Aubiquitin control probe (E) demonstrated the normalisation process hadbeen successful and that the RNA was intact.

FIG. 36 shows the effect of long-term culturing on pWE-TSN clones. Anumber of pWE-TSN mouse L-cell clones were grown continuously for 60generations. For freeze/thaw, clones were stored in liquid nitrogen forat least 2 days, defrosted and cultured for 1 week before RNA washarvested and the cells frozen for the next cycle. Experiments wereperformed with and without G418 present in the medium. TBP expressionwas assayed by using TB14 oligonucleotides and a human-specificrestriction endonuclease (as indicted by +) as described herein. Allsamples were analysed without the enzyme and were identical. Arepresentative (−) sample is also shown.

FIG. 37 shows analysis of TBP gene expression in pBL3-TPO-puro clones.The analysis for TBP gene expression was performed using the TB14primers with total RNA isolated from mouse L-cells transfected with thepBL3-TPO-puro construct as described herein. A (+) above a laneindicates that the PCR product has been digested with a human specificenzyme, (−) indicates no digestion (control). Human (K562) and mouse(non-transgenic lung) RNA controls are also shown as well as a no-RNAcontrol (dH₂O). Arrows indicate the positions of the uncut (human andmouse or mouse) and human specific products. Expression values arecorrected for copy number such that 100% expression means that a singlecopy of the transgene is expressing at the same level as one of the twoendogenous mouse genes. All copy numbers varied from 1-2 and areindicated above each bar.

FIG. 38 shows dot blot analysis of (B) human HP1 γ mRNA expression and(C) human hnRNP A2 mRNA. Tissue distribution of HP1 γ mRNA and hnRNP A2mRNA from within the hnRNP A2 cluster using a human multiple-tissue mRNAdot-blot: each segment is loaded with a given amount of poly(A)⁺ RNA (A,shown in ng below each tissue). The blot was hybridised with (B) a 717ntPCR fragment from the HP1 γ cDNA sequence and with (C) a 1237nt PCRprobe generated by using PCR primers 5′ GCTGAAGCGACTGAGTCCATG 3′ (SEQ IDNO:1) and 5′ CCAATCCATTGACAAAATGGGC 3′ (SEQ ID NO:2) for the expressionof hnRNP A2.

FIG. 39 shows the results of the FISH analysis of TBP transgeneintegrated into mouse Ltk cells demonstrating integration ontocentromeric heterochromatin. (A) shows a non-centromeric integration,(B) and (C) show two separate centromeric integrations.

FIG. 40 shows erythropoietin (EPO) expression in CHO cell pools stablytransfected with CET300 and CET301 constructs comprising the 7.5 kbsub-fragment located in the dual promoter regions between RNP andHP1H-γ, the CMV promoter and the gene encoding EPO.

FIG. 41 shows fluorescent EGFP expression of mouse Ltk cell clonestransfected with 16RNP-EGFP and its relationship to copy number. ClonesF1, G6 and I3 have 16RNP-EGFP co-localized with the murine centromericheterochromatin.

FIG. 42 shows the FISH analysis of mouse Ltk cells transfected with16RNP-EGFP. (A) shows clone H4 having a non-centromeric integration. (B,C, & D) show clones G6, F1 and I3 having centromeric integrations,respectively. t is the 16RNP-EGFP and c is the mouse centromere.

FIG. 43 shows FACS profiles of EGFP expression of HeLa cells transfectedwith EBV comprising 16RNP cultured in the presence of Hygromycin B overa period of 41 days.

FIG. 44 shows FACS profiles of EGFP expression of HeLa cells transfectedwith EBV comprising 16RNP cultured in the presence of Hygromycin Bthroughout and when Hygromycin B is removed from day 27.

FIG. 45 shows EPO production in cells transiently transfected withCET300, CET301 and CMV-EPO.

FIG. 46 shows results of ELISA detecting NTR expression for various AFPconstructs in HepG2 (AFP+ve) and KLN205 (AFP−ve) cells.

FIG. 47 shows NTR expression in HepG2 tumours and host mouse liversfollowing intratumoural injection with CTL102/CTL208.

FIG. 48 shows growth inhibition of HepG2 tumours following intratumouralinjection with CTL102/CTL208 and CB1954 administration.

FIG. 49 shows schematically the structure of vectors CET200 and CET210.

FIG. 50 shows the constructs generated and fragments used in comparisonto the hnRNP A2 endogenous genomic locus.

FIG. 51 shows a graph of the FACS analysis with median fluorescence ofHeLa populations transiently transfected with non-replicating plasmid.

FIG. 52 shows representative low magnification field of views of HeLacell populations transiently transfected with non-replicating plasmid.

DETAILED DECRIPTION OF THE INVENTION EXAMPLES Materials and Methods

Library Screening

Genomic clones spanning the human TBP and hnRNP A2 loci were isolatedfrom a P1-derived artificial chromosome (pCYPAC-2) library (CING-1;Ioannou et al., 1994). Screening was by polymerase chain reaction (PCR)of bacterial lysates.

Primers for TBP

Primers were designed using the partial genomic sequence described byChalut et al. (1995) and were as follows: TB3[5′ATGTGACAACAGTGCATGAACTGGGAGTGG3′] (SEQ ID NO:3) (−605) and TB4[5′CACTTCCTGTGTTTCCATAGGTAAGGAGGG3′] (SEQ ID NO:4) (−119) hybridise tothe 5′region (5′UTR) of the TBP gene and give rise to a 486 bp PCRproduct from the human gene only (see results). The numbers inparenthesis are with respect to the mRNA CAP site defined by Peterson etal., (1990).

TB5 [5′GGTGGTGTTGTGAGAAGATGGATGTTGAGG3′] (SEQ ID NO:5) (1343) and TB6[5′GCAATACTGGAGAGGTGGAATGTGTCTGGC3′] (SEQ ID NO:6) (1785) amplify aregion from the 3′UTR and produce a 415 bp product from both human andmouse DNA due to significant sequence homology in this region. Thenumbers in parenthesis are with respect to the cDNA sequence defined byPeterson et al., (1990).

Primers for hnRNP A2

Primers for hnRNP A2 were designed from the genomic sequence describedby Biamonti et al., (1994).

Hn1 [5′ ATTTCAAACTGCGCGACGTTTCTCACCGC3′] (SEQ ID NO:7) (−309) and Hn2[5′ CATTGATTTCAAACCCGTTACCTCC3′] (SEQ ID NO:8) (199) in the 5′ UTR togive a PCR product of 508 bp. Hn3 [5′ GGAAACTTTGGTGGTAGCAGGAACATGG3′](SEQ ID NO:9) (7568) and Hn4 [5′ ATCCATCCAGTCTTTTAAACAAGCAG 3′] (SEQ IDNO: 10) (8176) amplify a region in the penultimate exon (number 10) togive a PCR product of 607 bp. The numbers in parentheses are withrespect to the transcription start point defined by Biamonti et al.(1994).

PCR Protocol

PCR was carried out using 1 μl pooled clone material in a reactioncontaining 25 mM each dATP, dGTP, dCTP, dTTP, 1× reaction buffer (50 mMTris-HCl [pH 16 mM (NH₄)₂SO₄, 3.5 mM₂, 150 μg/ml bovine serum albumin),2.5 units Taq Supreme polymerase (Fermentas) and 1 μM each primer in atotal reaction volume of 25 μl. Cycling conditions were: 4 cycles of 94°C. for 1 minute, 62° C. for 1 minute, 72° C. for 1 minute, followed by30 cycles of 94° C. for 1 minute, 58° C. for 1 minute, 72° C. for 1minute. Positively identified clones were grown in T-Broth (12 gtryptone, 24 g yeast extract (both Difco), 23.1 g KH₂PO₄, 125.4 gK₂HPO₄, 0.4% glycerol per 1 litre distilled water; Tartof and Hobbs,1987) containing 30 μg/ml kanamycin. Permanent stocks of the bacteriawere prepared by freezing individual suspensions in 1× storage buffer(3.6 mM K₂HPO₄, 1.3 mM₂PO₄, 2.0sodium citrate, 1 mM MgSO₄, 4.4%glycerol) at −80° C.

CYPAC-2 DNA Isolation

Plasmid DNA was isolated using a modified alkaline lysis method(Birnboim and Doly, 1979), as follows. Baffled 2 liter glass flaskscontaining 1 litre T-broth were inoculated with a single bacterialcolony and incubated at 37° C. for 16 hours with constant agitation.Bacteria were harvested by centrifugation in a Beckman J6 centrifuge at4200 rpm (5020×g, similarly for all subsequent steps) for 10 minutes.Pellets were vortexed, re-suspended in 15 mM Tris[pH 8.0], 10 mM EDTA,10 μg/ml RNaseA (200 ml) and incubated at room temperature for 15minutes. Lysis solution (0.2M NaOH, 1% SDS; 200 ml) was added withgentle mixing for 2 minutes, followed by the addition of 200 mlneutralisation solution (3M potassium acetate [pH 5.5]) with gentlemixing for a further 5 minutes. Bacterial debris was allowed toprecipitate for 1 hour at 4° C. and then removed by centrifugation for15and filtration of the supernatant through sterile gauze. Isopropanol(400 ml; 40% final concentration) was added to precipitate the plasmidDNA at room temperature for 1 hour. After centrifugation for 15 minutesand washing of the pellet in 70% ethanol, the DNA was re-suspended in a4 ml solution of 1× TNE (50 mM Tris-HCl [pH 7.5], 5 mM EDTA, 100 mMNaCl), 0.1% SDS and 0.5 mg/ml Proteinase-K (Cambio) to remove residualproteins. Following incubation at 55° C. for 1 hour and subsequentphenol:chloroform (1:1 v/v) extraction, the DNA was precipitated with 1volume of 100% ethanol or isopropanol and spooled into 2 ml TE buffer(10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Yields of 50 μg/ml were routinelyobtained.

Restriction Enzyme Mapping

Restriction enzyme mapping was carried out by hybridisingoligonucleotides derived from both pCYPAC-2 and TBP gene sequences toSouthern blots (Southern, 1975) of restriction enzyme digested clonedDNA as described above. Oligonucleotides which hybridise to pCYPAC-2sequences just proximal to the Bam HI site into which genomic fragmentsare cloned were used, the sequences of whichwere:EY2:[5′(-TGCGGCCGCTAATACGACTCACTATAGG-3′ (SEQ IDNO:11)189:[5′(-GGCCAGGCGGCCGCCAGGCCTACCCACTAGTAATTCGGGA-3′(SEQ IDNO:12)E xcision of any genomic insert from pCYPAC-2 with Not I meansthat the released fragment will retain a small amount of plasmidsequence on each side. On the EY2 side this will be 30 bp with themajority of the EY2 sequence within the excised fragment. Hybridisationof this oligonucleotide to Not I digested pCYPAC-2 clones shouldtherefore, highlight the released genomic band on Southern blotanalysis. At the 189 side, the excised fragment will contain 39 bp ofplasmid sequence and the majority of the 189 oligonucleotide sequence is3′ to the Not I site, within pCYPAC-2. Therefore, this oligonucleotidewill hybridise to the vector on Not I digests of pCYPAC-2 clones.Approximately 100 ng plasmid DNA was subjected to restrictionendonuclease digestion using manufacturers recommended conditions(Fermentas), and subsequently electrophoresed on 0.7% agarose gels in0.5× TAE buffer (20 mM Tris-Acetate [pH 8.0], 1 mM EDTA, 0.5 μg/mlethidium bromide) or on pulsed field gels. Pulsed Field GelElectrophoresis (PFGE) was carried out on a CHEF-DRII system (Biorad) on1% PFGE agarose (FMC)/0.5× TAE gels at 6V/cm for 14 hours with switchtimes from 1 second to 30 seconds. Identical conditions were used forall PFGE analysis throughout this study. Gels were stained in 1 μg/mlethidium bromide solution before being photographed under ultravioletlight.

In preparation for Southern blot analysis, the DNA was depurinated byfirst exposing the agarose gels to 254 nm ultraviolet light (180,000μJ/cm² in a UVP crosslinker, UVP) and then subsequently denaturing bysoaking in 0.5M NaOH, 1.5M NaCl for 40 minutes with a change of solutionafter 20 minutes. The DNA was transferred to HYBOND-N nylon membrane(Amersham) by capillary action in a fresh volume of denaturationsolution for 16 hours. Crosslinking of the nucleic acids to the nylonwas achieved by exposure to 254 nm ultraviolet light at 120,000 μJ/cmMembranes were neutralised in 0.5M Tris-HCl [pH 7.5], 1.5M NaCl for 20minutes and rinsed in 2×SSC before use. (1×SSC is 150 mM NaCl, 15 mMsodium citrate, [pH 7.0]).

Oligonucleotide probes were 5′ end labelled with T4 polynucleotidekinase and ³²P-γ ATP to enable detection of specific fragments onSouthern blots. Each experiment employed 100 ng of oligonucleotidelabelled in a reaction containing 2 μl³²P-γ ATP (>4000 Ci/mmol; 10mCi/ml, Amersham) and 10 units T4 polynucleotide kinase (Fermentas) inthe manufacturers specified buffer. After incubation at 37° C. for2unincorporated nucleotides were removed by chromatography on SephadexG50 columns (Pharmacia) equilibrated with water. End-labelled probeswere typically labelled to a specific activity>1×10⁸ dpm/μg.

Hybridisation was carried with membranes sandwiched between nylon meshesinside glass bottles (Hybaid) containing 25 ml pre-warmed hybridisationmix (1 mM [pH 8.0], 0.25M Na₂HPO₄ [pH 7.2], 7% SDS; Church and Gilbert,1984) and 100 μg/ml denatured sheared salmon testis DNA. Afterpre-hybridisation at 65° C. for 1 hour, the solution was decanted andreplaced with an identical solution containing the labelled probe.Optimal hybridisation temperature was determined experimentally andfound to be 20° C. below the T_(m) for the oligonucleotide in TE buffer,calculated as T_(m)=59.9+41[% GC]−[675/primer length]). After 16 hourshybridisation membranes were removed and washed with three, 2 minuteswashes of 6×SSC, 0.1% SDS followed by exposure to x-ray film (BioMAX,Kodak).

DNA Constructs

A 44 kb genomic DNA region spanning the TBP gene with 12 kb of both 5′and 3′ flanking sequences, was derived from the pCP2pCYPAC-2 clone (seeFIG. 9) as a NotI fragment. This was cloned into the cosmid vector pWE15(Clontech) to generate pWE-TSN (FIG. 9). The vector exchange wasnecessary as the pCYPAC-2 plasmid does not contain a selectable markerfor eukaryotic cell transfection studies. Digestion of pCP2-TNN withNotI liberates a 44 kb fragment extending from the 5′ end of the genomicinsert to the NotI site present in the genomic sequence located 12 kbdownstream of the last exon of TBP (see FIG. 9). In addition, fragmentscontaining the remaining 20 kb of 3′ flanking sequence in this clone andthe pCYPAC-2 vector are produced. The ligation reaction was performedusing approximately 1 μg of NotI digested pCP2-TNN and 200 ng similarlycut pWE15 in a 10 μl reaction using conditions as described above. Afterheat inactivation of the T4 DNA ligase, the complete ligation mix waspackaged into infectious lambda>phage particles with Gigapack Gold III(Stratagene). Recombinant bacteriophage were stored in SM buffer (500 μlof 50 mM Tris-HCl, 100 mM NaCl, 8 mM MgSO₄, 0.01% (w/v) gelatine, 2%chloroform). Infection was carried out as follows: 5 ml of an overnightculture of E. coli DH5 α was centrifuged (3000×g, 5 minutes) and thebacteria resuspended in 2.5 ml of 10 mM MgCl₂. Equal volumes of packagedmaterial and E. coli were mixed and incubated at 25° C. for 15 minutesafter which time 200 μl was added and the mixture incubated at 37° C.for a further 45 minutes. The suspension was plated on LB-ampicillinagar plates and single colonies analysed as mini preparations thefollowing day. Large amounts of pWEwere prepared from 1 liter culturesas for pCYPAC-2 clones.

pCYPAC-2 DNA Sub-Cloning Methods

The following procedure was used in order to sub-clone small (less than10 kb) restriction enzyme fragments derived from pCYPAC-2 clones. DNAwas restriction enzyme digested and electrophoresed on 0.6% low meltingpoint agarose gels (FMC) with all ultraviolet photography carried out ata wavelength of 365 nm to minimise nicking of ethidium bromide stainedDNA (Hartman, 1991). The gel area containing fragments of the desiredrange of sizes was excised from the gel, melted at 68° C. for 10 minutesand allowed to equilibrate to 37° C. for a further 5 minutes. Theplasmid vector pBluescriptKS(+) (Stratagene) was similarly restrictionenzyme digested to give compatible termini with the pCYPAC-2 derivedDNA, treated with 10 units calf intestinal phosphatase (Fermentas) for 1hour to minimise selfand purified by phenol:chloroform (1:1 v/v)extraction followed by ethanol precipitation. Molten gel slices weremixed with 50 ng of this vector preparation giving a molar excess of 4:1fragment to vector molecules. T4 DNA Ligase (10 units; Fermentas) wasadded along with the specified buffer and the mixture incubated at 16°C. for 16 hours after which time the enzyme was heat inactivated (65° C.for 20 minutes) to improve transformation efficiency (Michelsen, 1995).Preparation of calcium chloride competent DH5α E. coli and subsequenttransformation was performed using established procedures (Sambrook etal., 1989). Transformation was achieved by melting and equilibrating theligation mixture to 37° C. before the addition of 100 μl competent cellsmaintaining a final agarose concentration of no more than 0.02%.Bacteria were incubated on ice for 2 hours followed by heat shock at 37°C. for 5 minutes and subsequent addition of 1 ml SOC media (20 gtryptone; 5 g yeast extract; 0.5 g NaCl; 20 mM glucose, [pH 7.0] per 1distilled water; Sambrook et al., 1989). After a further hour at 37° C.,cells were mixed with 50 μl selection solution (36 mg/ml Xgal, 0.1 MIPTG) and plated on the appropriate LB-antibiotic plates (10 g NaCl [pH7.0], 10 g tryptone, 5 g yeast extract, 20 g agar per liter distilledwater) containing 20 μg/ml ampicillin. After incubation at 37° C. for 16hours, bacterial colonies containing recombinant plasmids wereidentified by their white (as opposed to blue) colour due to disruptionof β-galactosidase gene activity. Selected colonies were analysed byrestriction digestion of DNA isolated from single colony minipreparations. Using this procedure it was possible to sub-clonefragments of up to 20 kb in size into the pBluescriptKS(+) vector.

FPCR amplified products were cloned using the following procedure. Aftera standard PCR reaction using 1 ng of the pCYPAC-2 derived clone DNA asa template in a 50 μl volume, 10 units T4 DNA polymerase (Fermentas)were added to the reaction and incubated for 30 minutes at 37° C. Afterinactivation of the polymerase enzyme (96° C., 20 minutes), 7 μl of thePCR product were ligated to 50 ng Eco RV digested pBluescriptKS(+)vector in a final volume of 10 μl. Use of the T4 DNA polymerase to bluntthe ends of the PCR products resulted in a high proportion ofrecombinant clones (data not shown).

Generation of pBL3-TPO-puro

pBL3-TPO-puro contains the entire 19 kb TBP gene with approximately 1.2kb 5=and 4.5 kb 3=flanking sequences and a puromycin resistance genecassette, sub-cloned into the pBL3 vector. This was achieved by 3consecutive cloning steps.

Firstly, the 4.5 kb of sequence flanking the 3′ end of the human TBPgene in the pCP2-TLN plasmid was sub-cloned from pCP2-TLN as a NotI BSaclI fragment. This fragment extends from the Sac lI site in the 3′ UTRof the TBP gene to the OL189-proximal NotI site within the pCYPAC-2vector. This fragment was cloned into SaclI and NotI digested pBL3 anddesignated MA426. The remaining TBP gene sequences reside on a 19 kbSaclI fragment extending from approximately 1.2 kb upstream of the mRNAcap site to the SaclI site in the 3′-UTR. This fragment was ligated into MA426 which was linearised with SaclI, and clones screened for thecorrect orientation.

DNA Sequencing and Computer Sequence Analysis

DNA was prepared using the Flexi-Prep system (Pharmacia) and automatedfluorescent sequencing provided as a service from BaseClear(Netherlands). dBEST and non-redundant Genbank databases were queriedusing previously described search tools (Altschul et al., 1997). Allexpressed sequence tag clones used in this study were obtained throughthe I.M.A.G.E. consortium (Lennon et al., 1996). Multiple sequencealignments and prediction of restriction enzyme digestion patterns ofknown DNA sequences was performed using the program PCGENE(Intelligenetics Inc., USA). Plots of CpG di-nucleotide frequency wereproduced using VectorNTI software (Informax Inc., USA).

Generation of Transgenic Animals

Preparation of TBP Fragments for Microinjection

The 90 kb genomic fragment (TLN) encompassing the TBP/PSMB1 gene regionwas isolated by NotI digestion of the pCP2-TLN clone and prepared formicroinjection using a modified sodium chloride gradient method (Dillonand Grosveld, 1993). Initially, bacterial lipopolysaccharide (LPS) wasremoved from a standard pCP2-TLN maxi preparation using an LPS removalkit (Quiagen) according to the manufacturer's instructions.Approximately 50 μg of DNA was then digested for 1 hour with 70 units ofNotI (Fermentas) and a small aliquot analysed by PFGE to check forcomplete digestion. A 14 ml 5-30% sodium chloride gradient in thepresence of 3 mM EDTA was prepared in ultra-clear centrifuge tubes(Beckman) using a commercial gradient former (Life Technologies). Thedigested DNA was layered on the top of the gradient using wide-borepipette tips to minimise shearing and the gradient centrifuged at 37,000rpm for 5.5 hours (at 251C) in a SW41TI swing-out rotor (Beckman).Fractions of approximately 300 μl were removed starting from the bottomof the gradient (highest density) into individual microcentrifuge tubescontaining 1 ml 80% ethanol followed by incubation at −20° C. for 1hour. DNA precipitates were collected by centrifugation at (14900×g, 15minutes). Pellets were washed in 70% ethanol, dissolved in 20 μltransgenic microinjection buffer (10 mM Tris-HCl [pH 7.4], 0.1 mM EDTA)and 5 μl aliquots from alternate fractions analysed by gelelectrophoresis to assess contamination of vector and chromosomal DNA.Those fractions, which appeared to be free of such contaminants, werepooled and the DNA concentration assessed by absorbance at 260 nm.

The 40 kb genomic fragment (TSN) was isolated from pWE-TSN by NotIdigestion and purification using electro-elution as previously described(Sambrook et al., 1989). After electro-elution, DNA was purified bysequential extraction with TE buffer-saturated phenol, phenol:chloroform(1:1 v/v) and twice with water saturated n butanol to remove residualethidium bromide. DNA was precipitated with 2 volumes of 100% ethanoland resuspended in microinjection buffer. Fragment integrity wasassessed by PFGE and concentration determined by absorbance at 260 nm.The 25 kb genomic fragment (TPO) was isolated from pBL3-TPO using anidentical procedure except the insert was liberated from the vector bydigestion with SalI.

Preparation of hnRNP A2 Fragments for Microinjection

The 160 kb genomic fragment (MA160) encompassing the hnRNP A2 generegion was isolated and prepared for microinjection by NruI digestion ofpCP2-HLN (FIG. 13A) and sodium chloride gradient ultracentrifugation asdescribed above.

The 60 kb genomic fragment (HSN; FIG. 13B) was isolated from MA160 byAat lI digestion and purification by PFGE as described above. The 60 kbband was excised from the gel and cut into slices. Each slice was meltedat 65° C. and 30 μl analysed by PFGE. The fraction showing the purestsample of the 60 kb fragment was retained. The melted gel volume wasmeasured, made 1× with Gelase buffer, equilibrated at 42° C. for 10minutes and 1 unit Gelase enzyme (Epicentre Technologies) added per 500μl. Samples were incubated overnight at 42° C. and then centrifuged for30 minutes at 4° C. The supernatant was decanted with a wide bore tipand drop-dialysed against 15 ml of transgenic microinjection buffer on a0.25 μm filter in a 10 cm Petri dish for 4 hours. The dialysed solutionwas transferred into a microcentrifuge tube and spun for 30 minutes at4° C. Fragment integrity was assessed by PFGE and concentrationdetermined by absorbance at 260 nm.

Generation of Transgenic Mice

Transgenic mice were produced by pronuclear injection of fertilised eggsof C57/B16 mice. Each DNA fragment was injected at a concentration of 1ng/μl in transgenic buffer.

This was performed as a service by the UMDS Transgenic Unit (St Thomas'sHospital, London) using standard technology. Transgenic founders wereidentified using PCR screening of tail biopsy DNA isolated as follows.Approximately 0.5 cm tail biopsies from 10-15 day old mice wereincubated at 37° C. for 16 hours in 500 μl tail buffer (50 mM Tris-HCl[pH 8.0], 0.1M EDTA, 0.1M NaCl, 1% SDS, 0.5 mg/ml Proteinase-K). Thehydrosylate was extracted by gentle inversion with an equal volumephenol:chloroform (1:1 v/v) followed by centrifugation (14900×g, 15minutes). The DNA was precipitated from the aqueous phase by theaddition of 2 volumes of 100% ethanol and washed in 70% ethanol. DNA wasspooled and dissolved in 100 μl TE buffer. Typically, 50-200 μg DNA wasobtained as determined by absorbance measurements at 260 nm. Theconditions for the PCR reactions were as described for the screening ofthe pCYPAC-2 library using 100 ng tail biopsy DNA as template and theTB3/TB4 primer set. Positive founders were bred by back-crossing towild-type C57/B16 mice to generate fully transgenic F1 offspring.

Transgene Integrity and Copy Number Integrity and Copy Number

Transgene copy number and integrity was assessed by Southern blotanalysis of Bam HI, BglII, Eco RI, and HindIII digested tail biopsy DNA.Approximately 10 μg DNA was digested with 20-30 units of the specificrestriction endonuclease and electrophoresed on 0.7% agarose/0.5× TBE(45 mM Tris-borate, [pH 8.0], 1 mM EDTA,) gels for 16 hours at 1.5V/cm.Staining and transfer of DNA onto nylon membranes was as for plasmidSouthern blots except a positively charged matrix (HYBOND N+, Amersham)was used.

DNA probes were prepared by restriction enzyme digestion to remove anycloning vector sequences and purified from low-melting point agaroseusing the Gene-Clean system (Bio101, USA). Radioactive labelling of 100ng samples of the probes was performed by nick translation using acommercially available kit (Amersham) and 200 pmol each of dCTP, dGTP,dTTP and 3 μl α-P³²-dATP (specific activity>3000 Ci/mmol, 10 mCi/ml,Amersham). The enzyme solution consisting of 0.5 units DNA polymeraseI/10 pg DNase I in a standard buffer, was added and the reactionincubated at 15° C. for 2.5 hours. Probes were purified by Sephadex G-50chromatography and boiled for 5immediately prior to their use.Typically, specific activities of >1×10⁸ cpm/μg were obtained.

Hybridisation was performed as for plasmid Southern blots describedabove. Membranes were incubated in 15 ml pre-hybridisation solution(3×SSC, 0.1% SDS, 5× Denhardt's solution [100× Denhardt's solution is 2%Ficoll (Type 400, Pharmacia), 2% polyvinyl pyrollidone, 2% bovine serumalbumin (Fraction V, Sigma) per litre distilled water]), containing 100μg/ml denatured salmon testis DNA at 65° C. for 1 hour. The solution wasthen replaced by 15 ml hybridisation solution (as pre-hybridisationsolution with the addition of dextran sulphate to 10%) containing 100μg/ml denatured salmon testis DNA and the heat denatured radio-labelledprobe. After hybridisation at 65° C. for 16 hours membranes were washedthree times in 2×SSC/0.1% SDS for 30 minutes each and exposed toPhosphorImager (Molecular Dynamics) screens or x-ray film at −80° C.Those blots which were to be re-analysed, bound probe was removed bysoaking in 0.2M NaOH for 20 minutes followed by neutralisation asdescribed above.

The majority of the probes used in this study were derived from regionsof the genomic clones where no sequence information was available (e.g.pCP2-TLN end-fragment probes and those derived from the TBP intronicregions). A number of probes hybridised non-specifically to humangenomic DNA suggesting the presence of repetitive sequence elements. Inorder to circumvent this problem, aliquots of probe DNA wereindividually digested with a number of restriction enzymes,electrophoresed and Southern blotted. Enzymes with short recognitionsites (which should occur very frequently within the DNA), were chosenso as to digest the probe into a number of smaller fragments.Radiolabelled human C₀t−1 DNA was used as a probe to indicate thosefragments that contained repetitive sequences. Using this procedure, itwas possible to obtain fragments>500 bp that did not hybridise to theC₀t−1 probe, for all probes which contained repetitive elements.

Preparation of Cosmid DNA and Generation of Single Copy L-cell Clones

pWE-TSN DNA was prepared by alkaline lysis of 1 litre cultures asdescribed above until the isopropanol precipitation stage. Afterincubation at 25° C. for 1 hour, the pellet was resuspended in 300 μl TEand then added with continuous mixing to 10 ml Sephaglas FP DNA bindingmatrix (Pharmacia). The solution was constantly inverted for 10 minutesand the martix-bound DNA collected by centrifugation (280×g, 1 minute).The pellet was washed firstly with WS buffer (20 mM Tris-HCl [pH 7.5], 2mM EDTA, 60% ethanol), collected by centrifugation, washed with 70%ethanol and re-centrifuged. DNA was eluted from the matrix byresuspending the pellet in 2 ml TE buffer and incubation at 70° C. for10 minutes with periodic mixing. The solution was centrifuged (1100× g,2 minutes) and the DNA containing supernatant split equally into twomicrofuge tubes. Residual Sephagiass was removed by centrifugation(14950× g, 15 minutes), the supernatants pooled and DNA precipitatedwith 2 volumes of ethanol. The spooled DNA was washed once in 70%ethanol and resuspended at 1 μg/μl in sterile water. Approximately75-100 μg of pure cosmid DNA was obtained using this procedure, whichrepresents a yield of 60-80% of DNA obtained without Sephaglaspurification.

Transfection of adherent mouse L-cells (Earle et al., 1943) wasperformed as follows. Approximately 1×10⁷ cells grown in DMEM containing10% heat inactivated foetal calf serum (PAA laboratories), 2 mML-glutamine, were mixed with 1 μg pWE-TSN DNA linearised with SalI andincubated on ice for 10 minutes. DNA was introduced into the cells byelectroporation (Chu et al., 1987) with settings of 960 μF, 250V in aBiorad Gene-Pulser. Transfected cells were selected for and maintainedin the same medium including 400 μg/ml geneticin sulphate (G418; LifeTechnologies Inc.). Individual clones were isolated using cloning rings(Freshney, 1994). Thick-walled stainless steel cloning rings (LifeTechnologies Inc.) were autoclaved in silicon grease and transferred tothe tissue culture plate such that the colony was isolated. A solutionof trypsin (300 μl of 0.25% trypsin [pH 7.6] (Difco), 0.25M Tris-HCl [pH8.0], 0.4% EDTA [pH 7.6], 0.12M NaCl, 5 mM glucose, 2.4 mM KH₂PO₄ 0.84mM Na₂HPO₄.12H₂O, 1% phenol red) was added and the plate incubated at37° C. for 5 minutes. Cells were transferred to 24 well plates andclonal cell lines established. Clones were preserved as follows.Approximately 1×10⁷ cells were harvested by centrifugation, resuspendedin 0.75 ml freezing mix (70% standard growth media but including 20%foetal calf serum and 10% DMSO) and snap frozen on dry ice for 1 hourbefore to liquid nitrogen storage.

Genomic DNA was prepared from these L-cell clones using standardprocedures (Sambrook et al., 1989). Cells in T75 flasks were grown toconfluency (approximately 4×10⁷), the media removed and the flask washedwith PBS (2.68 mM KCl, 1.47 mM KH₂PO₄, 0.51 mM MgCl₂, 136.89 mM NaCl,8.1 mM Na₂HPO₄ [pH 7.3]) and 2 ml lysis buffer (10 mM Tris-HCl [pH 7.5],10 mM EDTA, 10 mM NaCl, 0.5% SDS, 1 mg/ml Proteinase-K) added. Cellswere dislodged from the culture flask by scraping and transferred to a15 ml centrifuge tube using a wide bore pipette tip. Lysis was allowedto proceed at 68° C. for 16 hours after which the solution was extractedonce with phenol:chloroform (1:1 v/v) and the DNA precipitated with anequal volume of isopropanol. After washing in 70% ethanol, the DNA wasresuspended in 1 ml TE buffer and concentration assessed by absorbanceat 260 nm.

Transfected gene copy-numbers were determined by Southern Blot analysisof BgIII digested genomic DNA. Human TBP was detected using a specificprobe (1.4H×) located in the C5 gene, 4 kb 5′ of the TBP transcriptioninitiation region and which detects a 4.2 kb fragment (see FIG. 10). Inaddition, blots were simultaneously probed with a 1 NcoI fragmentderived from the endogenous murine vav locus (Ogilvy et al., 1998) thatgives a 5.2 kb band and that acts as a single copy reference standard.Human TBP transgene copy-number was ascertained by comparing the ratioof the TBP to vav signal obtained with the 3 copy transgenic mouse lineTLN:8 after analysis of blots by PhosphorImager.

Total RNA was prepared from approximately 4×10⁷ cells by selectiveprecipitation in 1 ml of 3M LiCl, 6M urea (Auffrey and Rougeon, 1980;see Antoniou, 1991).

DNase I Hypersensitive Site Analysis

This was performed as previously described (Forrester et al., 1987;Reitmann et al., 1993). Nuclei were prepared from approximately 1×10⁹K562 cells (Lozzio and Lozzio, 1975). Harvested cells were washed in PBSand resuspended in 4 ml ice cold RSB (10 mM Tris-HCl [pH 7.5], 10 mMNaCl, 3 mM₂) and placed in a glass dounce homogeniser fitted with aloose pestle. After the addition of 1 ml of 0.5% NP40/RSB cells werehomogenised slowly for 10-20 strokes and nuclei recovered by theaddition of 50 ml RSB and centrifugation at 4° C. (640×g, 5 minutes).The supernatant was discarded and nuclei were resuspended in 1 ml RSBwith 1 mM CaCl₂. Immediately, a 100 μl aliquot (representingapproximately 1×10⁸ nuclei) was taken and DNA purified as describedbelow, to control for endogenous nuclease activity during the isolationprocedure.

The DNase I digestion was performed as follows. A range of aliquots (0,0.5, 1, 2, 3, 4, 5, 6, 8, 10 μl) of 0.2 mg/ml DNase I (Worthington) wasadded to individual microfuge tubes containing 100 μl of nuclei andincubated at 37° C. for 4 minutes. The digestion was stopped by theaddition of 100 μl 12× stop mix (20 mM[pH 8.0], 10 mM EDTA, 600 mM NaCl,1% SDS), 10 μl Proteinase-K (10 mg/ml concentration) and incubation at55° C. for 60 minutes. DNA was purified by phenol:chloroform (1:1 v/v)extraction and ethanol precipitation. Samples were electrophoresed on0.7% agarose/0.5× TBE gels and Southern blotted for analysis using³²P-radiolabelled probes.

RNA Preparation

Adult mice aged 10-40 weeks were sacrificed by cervical dislocation andwhole tissues isolated, snap frozen in liquid nitrogen and stored at−80° C. until required. Total RNA was prepared by selectiveprecipitation in 3M LiCl, 6M urea (Auffray and Rougeon, 1980). Tissueswere transferred to 14 ml tubes containing 1 ml of the LiClsolution andhomogenised for 30 seconds with an Ultra-Turrax T25 Janke & Kunkel).Samples were then subjected to three, 30-second pulses of sonication(Cole-Parmer Instrument Co., USA), the homogenate transferred to sterilemicrofuge tubes and RNA allowed to precipitate at 4° C. for 16 hours.The RNA was collected by centrifugation (4° C., 14900×g, 20 minutes)washed in 500 μl LiClsolution and resuspended in 500 μl TES (10 mMTris-HCl [pH 7.5], 1 mM EDTA, 0.5% SDS). After extraction withphenol:chloroform, samples were made 0.3M with sodium acetate and RNAprecipitated by the addition of 1 ml 100% ethanol and storage at −20° C.for at least 1 hour. The RNA was collected by centrifugation andresuspended in 20 μl sterile water and concentration assessed byabsorbance at 260 nm.

Competitive RT-PCR Based Assay

Analysis of Human TBP Expression

A modified competitive RT-PCR approach (Gilliland et al., 1990) was usedto accurately quantify human TBP and PSMB1 gene expression in a mousebackground. Total RNA (1 μg) from transgenic mouse tissues or cell lineswas reversed transcribed in a 25 μl reaction consisting of 10 unitsAvian Myeloblastosis Virus (AMV) reverse transcriptase (Promega), 10 mMDTT, 2.5 mM each dNTP, 25 units ribonuclease inhibitor (Fermentas) with1 μM reverse primer (TB14 or C5R) in 1× RT buffer (25 mM Tris-HCl [pH8.3], 25 mM KCl, 5 mM MgCl₂, 5 mM DTT, 0.25 mM spermidine). Synthesis ofcDNA was allowed to proceed at 42° C. for 1 hour followed by a furtherhour at 52° C. and heat inactivation of the enzyme at 95° C. for 5minutes. PCR reactions contained 1 μl cDNA amplified using the reactionmix described for tail biopsy screening and containing specific primersets for the sequence in question (as detailed above, one of which wasend-labelled using the protocol described above. Primers were purifiedwith two rounds of Sephadex-G25 chromatography (Pharmacia) and an 80%recovery was assumed. PCR conditions were 94° C. for 1 minute, 58° C.for 1 minute and 72° C. for 1 minute with cycle numbers between 5 and30.

In order to distinguish between human and mouse PCR products, 2-10 μl ofeach sample was incubated with 5 units of the appropriate restrictionenzyme at 37° C. for 2 hours. This reaction was carried out in a large(250 μl) volume to dilute salts and detergents from the PCR buffer toprevent inhibition of restriction enzyme activity. (Control experimentsdemonstrated that this was indeed the case). Digested and undigestedsamples were ethanol precipitated in the presence of 25 μg yeast tRNA(Sigma) as co-precipitant, collected by centrifugation and resuspendedin 5 μl gel loading buffer (5 mM Tris-Borate [pH 8.3], 1 mM EDTA, 7MUrea, 0.1% xylene cyanol, 0.1% bromophenol blue). Samples were analysedon pre-run, 5% polyacrylamide gels in the presence of 7M Urea (NationalDiagnostics) as denaturant and 0.5× TBE buffer. After electrophoresis at40V/cm for 1 hour, the gel was cut to remove residual unincorporatednucleotide running below the xylene cyanol dye front, dried and exposedto x-ray film or PhosphorImager screens.

Analysis of Human hnRNP A2 Expression

A similar competitive RT-PCR approach (Gilliland et al., 1990) was usedto accurately quantify human hnRNP A2 gene expression in a mousebackground. After reverse transcription, cDNA samples were amplified byPCR using primer sets Hn9 and Hn12 [5′-CTCCACCATATGGTCCCC-3′] (SEQ IDNO:13), one of which was end-labelled using the protocol describedabove. In order to distinguish between human and mouse hnRNP A2 PCRproducts, 2-10 μl of each sample was digested with 5 units HindIII at37° C. for 2 hours, purified, resolved on 5% denaturing polyacrylamidegels and results quantified as described above.

Sequencing and Bioinformatic Analyses of Clones

HindIII genomic clones of both TBP (nucleotides 1-9098, FIG. 20) andhnRNP A2 (nucleotides 1-15071, FIG. 21) loci were sequenced byBaseclear, Leiden, N L. Using a primerwalking strategy starting withprimers made to known sequence, regions of unknown sequence weregenerated; TBP nucleotides 1-5642 and hnRNPA2 nucleotides 1-3686.

These sequences were spliced together with previously known sequencedata and were then used in bioinformatic analyses.

Direct comparisons were made between TBP and hnRNPA2 sequences usingstandard Smith-Waterman searching. This showed no obvious regions ofhomology other than several Alu repeats as shown in FIG. 19. Maskingthese repeats and performing a comparison using the GCG bestfit programresulted in two short regions of homology as follows:

RNP 3868-3836: TBP 8971-9003 length=33% identity=75.758

RNP 3425-3459: TBP 9049-9083 length=35% identity=74.286

CpG-islands were also identified and are shown in FIG. 19. Nucleotidepositions are as follows:

RNP 4399-5491, 5749-6731

TBP 5285-5648, 6390-6966

Sequencing studies were performed as described above so as to providemore sequence data from the region immediately upstream of the RNP andTBP genes.

The sequence data given in FIGS. 20 and 21 begins at the 5′ HindIII siteand includes the Baseclear generated sequence and the already publishedsequence data spliced together. In the case of the TBP sequence theBaseclear sequence is denoted in capitals.

Analysis of these sequences demonstrated the existence of a previouslycharacterised gene, HP1H-γ, or heterochromatin associated proteinH-gamma upstream of the RNP gene (FIGS. 19 and 22). This gene has alsobeen shown to be ubiquitously expressed by human tissue dot blotanalysis (data not shown).

Bioinformatic analysis and sequence comparisons showed no obvioussequence homologies between the loci. However, a summary of the data isshown in FIG. 19. As can be seen, several putative Sp1 transcriptionfactor binding sites are located in the bidirectional promoter regionsof the two loci. The CpG methylation free islands are also indicated.Both loci show a bidirectional structure containing a cluster ofubiquitously expressed genes.

Construction of hnRNP A2 EGFP Reporter Constructs

CMV-EGFP-IRES was constructed by digesting pEGFP-N1 (Clontech) with KpnIand NotI to liberate the EGFP sequence, this was then ligated intoplRESneo (Clontech) that had been partially digested with KpnI and thenNotI. This created a vector with the EGFP gene 3′ to the CMV promoterand 5′ to IRESneo (CMV-EGFP-IRES).

The CMV promoter was exchanged for the RNP promoter to create theconstruct referred to in FIG. 22 as RNP. CMV EGFP-IRES was digested withAgeI, blunted with T4 DNA polymerase (50 mM Tris pH 7.5, 0.05 mM MgCl₂,0.05 mM DTT, 1 mM dNTP, 1 u T4 DNA polymerase/μg DNA) and then cut withNruI to release the CMV promoter to give EGFP-IRES. The RNP promoter wasremoved from an 8 kb hnRNPA2 HindIII clone (8 kb Hind BKS) whichcontained the promoters and first exons of the RNPA2 and HP1H-γ genes. 8kb Hind BKS was cut with BspEI and Tth111I (to release the 630 bppromoter) blunted with T4 DNA polymerase, and the isolated RNP promoterligated into EGFP-IRES.

5.5RNP was constructed by inserting the EGFP-IRES cassette into 8 kbHind BKS such that expression of EGFP was under the control of the RNPpromoter. The latter was partially digested with Tth111I, blunted withT4 DNA polymerase and then digested with SalI, this removed allsequences 3′ to the RNP promoter. The EGFP-IRES cassette was removedfrom CMV-EGFP-IRES by digestion with AgeI and blunted prior to digestionwith XhoI. This was then ligated into the restricted 8 kb Hind BKS.

5.5CMV was constructed by inserting the CMV-EGFP-IRES cassette into 8 kbHind BKS with the subsequent removal of the RNP promoter. 8 kb Hind BKSwas cut with BspEI, blunted and then digested with SalI removing the RNPpromoter and all sequences 3′ to the promoter. The CMV-EGFP-IREScassette was removed from CMV-EGFP-IRES by digestion with NruI and XhoIand ligated into the digested 8 kb Hind BKS.

Approximately 4 kb of DNA was removed from 5.5 RNP to leave 1.5 kb 5′ tothe RNP promoter creating 1.5RNP. This was achieved by digesting 5.5 RNPwith BamHI which gave fragments of 4, 2.9 and 5 kb. The 2.9 and 5 kbfragments were then isolated and religated to create 1.5 RNP, when the2.9 kb fragment was inserted in the correct orientation.

The 5.5RNP construct was extended to include hnRNPA2 sequences 3′ to theRNP promoter (constructs 7.5RNP and 8.5RNP), this region included thefirst exon and intron of hnRNPA2. In order to include the EGFP-IRESreporter in these constructs it was necessary to place the hnRNPA2splice acceptor sequence of exon 2 in frame with the EGFP gene such thatthe first exon of hnRNPA2 could splice to the EGFP gene and hence EGFPexpression could be driven off the RNP promoter. Two constructs weremade which included the hnRNPA2 splice acceptor, these contained 80 bpand approximately 1 kb of sequence 5′ to the second exon, thesesequences were obtained by PCR from MA160 which includes the wholehnRNPA2 genomic sequence. The 80 bp sequence was isolated by PCR (20mMTris-HCl pH 8.4, 50 mM KCl, 1 μM Primer, 2 mM MgCl₂, 0.2 mM dNTP 3.5μg MA160 DNA, 5U Platinum Taq DNA Polymerase) using primers[5′ACCGGTTCTCTCTGCAAAGGAAAATACC 3′] (SEQ ID NO:14) and[5′GGTACCCTCTGCCAGCAGGTCACCTC 3′] (SEQ ID NO:15), the 1 kb fragment wasisolated using the primers [5′ACCGGTTCTCTCTGCAAAGGAAAATACC 3′] (SEQ IDNO:16) and [5′GGTACCGAGCATGCGAATGGAGGGAGAGCTCCG 3′](SEQ ID NO:17). Theprimers were designed such that the PCR product contained KpnI and AgeIsites at the 5′ and 3′ ends respectively. PCR products were then clonedinto the TA cloning vector pCR3.1 (Invitrogen).

The 80 bp and 1 kb fragments were isolated from pCR3.1 as KpnI-AgeIfragments and ligated into CMV-EGFP-IRES that had been partiallydigested with KpnI and then cut with AgeI, this created inframe fusionsof the splice acceptor (SA) with the EGFP gene.

7.5RNP was constructed by digesting 8 kb Hind BKS with ClaI, bluntingwith T4 DNA polymerase, then digesting with SalI. The 80 bp SA-EGFP-IREScassette was isolated by a KpnI partial digest followed by blunting withT4 DNA polymerase and XhoI digestion. This was ligated into theClaI-SalI digested 8 kb Hind BKS.

8.5RNP was constructed by an SphI partial digest of 8 kb Hind BKSfollowed by digestion with SalI, the 1 kb SA-EGFP-IRES cassette wassimilarly isolated by an SphI partial digest followed by restrictionwith XhoI. The cassette was ligated into 8 kb Hind BKS to create 8.5RNP.

4.0CMV was constructed by excising a 4 kb fragment from 8 kb Hind BKSwith Bam HI/HindIII/BstEII digestion. The ends of the fragment were thenend-filled with Klenow and T4 DNA polymerase.

pEGFP-N1 (Clontech) was linearised with AseI, the ends blunted as aboveand then treated with calf intestinal phosphatase (CIP). Both fragmentswere then ligated overnight.

p7.5CMV was constructed by excising the 8.3 kb fragment from p8 kb HindBKS with HindIII digestion. The ends of the fragment were then endfilled with Klenow and T4 DNA Polymerase. pEGFP-NI (Clontech) waslinearised with AseI, the ends were blunted as above and then treatedwith calf intestinal phosphatase (CIP). Both fragments were then ligatedovernight. The resultant clones were screened for both forward andreverse orientations of the 8.3 kb UCOE insert

p16CMV was constructed by excising a 16 kb fragment from MA551 (hnRNPA2genomic clone containing 5 kb 5′ and 1.5 kb 3′ sequence including theentire coding region (16 kb fragment shown in FIG. 13C)) by SalIdigestion. The ends of the fragment were then end filled with Klenow andT4 DNA Polymerase. pEGFP-NI (Clontech) was linearised with AseI, theends were blunted as above and then treated with calf intestinalphosphatase (CIP). Both fragments were then ligated overnight. Theresultant clones were screened for both forward and reverse orientationsof the 16 kb UCOE insert.

CHO Transfection

CHO cells were harvested at 2×10⁷ cells/ml in serum free medium. 1×10⁷cells (0.5 ml) were used per transfection, along with 1 ug (5 ul) oflinear DNA and 50 ug (5 ul) of salmon sperm carrier DNA. The DNA andcells were mixed and left on ice for 10 minutes. Cells wereelectroporated using the BioRad Gene Pulser II™ at 975 uF/250V and thenleft on ice for 10 minutes. The mix is then layered onto 10 mls ofcomplete medium (HF10) and spun at 1400 rpm for 5 minutes. Thesupernatant is removed and the pellet resuspended in 5 mls of HF10. Thecells were then plated out at 5×10⁴ or 1×10⁴ in 10 cm dishes and at2×10⁶ cells per T225 flask. After 24 hrs the cells were placed underselection, initially at 300 ug/ml G418 and then after 4 days at 600ug/ml G418. 10 days after transfection colonies were stained withmethylene blue (2% solution made up in 50% ethanol) and counted.Duplicate plates were maintained in culture either as restricted poolsor as single cell clones.

Analysis of GFP Expression in Transfected CHO Clones

The transfected cells were maintained on G418 selection at 600 μg/ml.Cells were stripped off 6-well plates for expression analysis of GFP.Cells were washed with phosphate buffered saline (PBS; Gibco) andincubated in Trypsin/EDTA (Sigma) until they had detached from thesurface of the plates. An excess of Nutrient mixture F12 (HAM) medium(Gibco) supplemented with 10% foetal calf serum (FCS; Sigma) was addedto the cells and the cells transferred to 5 ml polystyrene round-bottomtubes. The cells were then analysed on a Becton-Dickinson FACScan forthe detection of GFP expression in comparison to the autofluorescence ofthe parental cell population. 19 RNP clones, 24 5.5RNP clones, 21 CMVclones and 12 5.5CMV clones were analysed and the average taken of themedian fluorescence of all the positive clones.

Analysis of GFP Expression in Transfected CHO Pools

Colonies of transfected CHO cells, that had undergone selection on G418,were stripped from a T225 tissue culture flask and plated on 10 cm petridishes to give approximately 100 colonies/plate. When the colonies hadgrown up, the cells were stripped and this limited pool of transfectedcells was analysed for GFP expression. GFP expression was monitored on aregular basis, with the pools split 1:10 every 3-4 days. Cells werealways split into 24-well plates the day before analysis, so that thecells were approximately 50% confluent on the day of analysis. The cellswere then stripped from the 24-well plates and analysed in the same wayas the previous section. For the expression time course, a marker region(M1) was set which contained only a minor proportion of the positivepopulation of cells and was used to investigate any loss of GFPexpression from the initial level over time.

FISH Analysis of Single/Low Copy Number Integrants

FISH analysis using the 40 kb TBP cosmid pWE-TSN or the pBL3-TPO-puro.

FMouse Ltk—cells grown in DMEM—10% fetal calf serum were electroporatedwith the 40 kb TBP cosmid pWE-TSN (FIG. 9) or the 25 kb plasmidpBL3-TPO-puro. The transfectants were selected with either 200 mg/mlG418 (TSN) or 5 mg/ml puromycin (TPO) and single or low copy clones weregenerated as outlined previously. Logarithmically growing cells from theselected clones were treated with 0.4 mg/ml colchicine for 1 h prior toharvest. Cells were then hypotonically swollen in 0.056 M KCl, fixed in3:1 methanol-acetic acid, and spread on microscope slides to obtainmetaphase chromosomes. The slides were pretreated with 100 mg ofRNaseA/ml in 2×SSC (1×SSC is 0.15 M NaCl, 0.015 M sodium citrate) for 1h at 37° C., washed in 2×SSC, and put through an ethanol dehydrationseries (70, 90, and 100% ethanol). The chromosomes were denatured at 70°C. for 5 min in 70% formamide-2×SSC, plunged into ice-cold 70% ethanol,and dehydrated as before. One hundred nanograms of TBP probe (entire TPOplasmid carrying 25 kb of human genomic DNA comprising the TBP gene) and50 nanograms of mouse gamma-satellite probe (as described by Horz etal., Nucl. Acids Res. 9; 683-696, 1981) were labelled withdigoxigenin-11-dUTP and biotin-16-dUTP, respectively, by nicktranslation (Boehringer) following manufacturer=s instructions. Labelledprobes were precipitated with 1 mg of cot-1 DNA and 5 mg of herringsperm DNA, resuspended in 50% formamide-2×SSC-1% Tween 20-10% dextransulfate, denatured at 75° C., the TBP probe preannealed for 30 min at37° C. and pooled and applied to the slides. Hybridization was carriedout overnight at 37° C. The slides were washed four times for 3 min eachtime in 50% formamide-2×SSC at 45° C., four times for 3 min each time in2×SSC at 45° C., and four times for 3 min each time in 0.1×SSC at 60° C.After being washed for 5 min in 4×SSC-0.1% Tween 20, the slides wereblocked for 5 min in 4×SSC-5% low-fat skimmed milk. The biotin labelledprobe was detected by 30 min incubation at 37° C. with each of thefollowing: avidin-conjugated Texas Red (Vector Laboratories Inc, USA)followed by biotinylated anti-avidin (Vector Laboratories Inc, USA) andavidin-conjugated Texas Red (Vector Laboratories Inc, USA). Digoxigeninlabelled probe was detected at the same time as biotin detection witheach of the following: anti-digoxigenin-fluorescein (FITC, Boehringer)followed by mouse anti-FITC (DAKO) and horse fluorescein-conjugated antimouse IgG (Vector Laboratories Inc, USA). Between every two incubations,the slides were washed three times for 2 min each time in 4×SSC-0.1%Tween 20. The slides were counterstained with DAPI(4=−6-diamidino-2-phenylindole) and mounted in Vectashield (VectorLaboratories Inc, USA). Images were examined with an oil 100× objectiveon a fluorescence microscope. The images were capture using aPhotometrics cooled charge-couple device camera and Vysis Smartcapturesoftware

FISH Analysis Using the 16RNP-EGFP Construct.

The 6RNP-EGFP vector was constructed by inserting the E Mouse Ltk-cellsgrown in DMEM-10% fetal calf serum were electroporated with the 40 kbTBP cosmid pWE-TSN (FIG. 9) or the 25 kb plasmid pBL3-TPO-puro. Thetransfectants were selected with either 200 mg/ml G418 (TSN) or 5 mg/mlpuromycin (TPO) and single or low copy clones were generated as outlinedpreviously. Logarithmically growing cells from the selected clones weretreated with 0.4 mg/ml colchicine for 1 h prior to harvest. Cells werethen hypotonically swollen in 0.056 M KCl, fixed in 3:1 methanol-aceticacid, and spread on microscope slides to obtain metaphase chromosomes.The slides were pretreated with 100 mg of RNaseA/ml in 2×SSC (1×SSC is0.15 M NaCl, 0.015 M sodium citrate) for 1 h at 37° C., washed in 2×SSC,and put through an ethanol dehydration series (70, 90, and 100%ethanol). The chromosomes were denatured at 70° C. for 5 min in 70%formamide-2×SSC, plunged into ice-cold 70% ethanol, and dehydrated asbefore. One hundred nanograms of TBP probe (entire TPO plasmid carrying25 kb of human genomic DNA comprising the TBP gene) and 50 nanograms ofmouse gamma-satellite probe (as described by Horz et al., Nucl. AcidsRes. 9; 683-696, 1981) were labelled with digoxigenin-11-dUTP andbiotin-16-dUTP, respectively, by nick translation (Boehringer) followingmanufacturer=s instructions. Labelled probes were precipitated with 1 mgof cot-1 DNA and 5 mg of herring sperm DNA, resuspended in 50%formamide-2×SSC-1% Tween 20-10% dextran sulfate, denatured at 75° C.,the TBP probe preannealed for 30 min at 37° C. and pooled and applied tothe slides. Hybridization was carried out overnight at 37° C. The slideswere washed four times for 3 min each time in 50% formamide-2×SSC at 45°C. four times for 3 min each time in 2×SSC at 45° C. and four times for3 min each time in 0.1×SSC at 60° C. After being washed for 5 min in4×SSC-0.1% Tween 20, the slides were blocked for 5 min in 4×SSC-5%low-fat skimmed milk. The biotin labelled probe was detected by 30 minincubation at 37° C. with each of the following: avidin-conjugated TexasRed (Vector Laboratories Inc, USA) followed by biotinylated anti-avidin(Vector Laboratories Inc, USA) and avidin-conjugated Texas Red (VectorLaboratories Inc, USA). Digoxigenin labelled probe was detected at thesame time as biotin detection with each of the following:anti-digoxigenin-fluorescein (FITC, Boehringer) followed by mouseanti-FITC (DAKO) and horse fluorescein-conjugated anti mouse IgG (VectorLaboratories Inc, USA). Between every two incubations, the slides werewashed three times for 2 min each time in 4×SSC-0.1% Tween 20. Theslides were counterstained with DAPI (4=−6-diamidino-2-phenylindole) andmounted in Vectashield (Vector Laboratories Inc, USA). Images wereexamined with an oil 100× objective on a fluorescence microscope. Theimages were capture using a Photometrics cooled charge-couple devicecamera and Vysis Smartcapture software. GFP-IresNeo expression cassetteand some RNP 5′ sequences from 8.5RNP into MA551. 8.5 RNP was digestedwith Xho I, blunted with T4 DNA polymerase and then digested with Pac I,the resulting fragment was ligated into MA551 that had been cut with NheI, blunted and then digested with PacI. As with 8.5RNP expression isdriven off the RNP promoter resulting in an in-frame fusion of exon 1 ofRNP with EGFP.

Clones of mouse LTK cells transfected with 16RNP-EGFP were grown inDMEM-10% fetal calf serum and 200 μg/ml G418. Logarithmically growingcells were treated with 0.4 g/ml colchicine for 1 h prior to harvest.Cells were hypotonically swollen in 0.056 M KCl, fixed in 3:1methanol-acetic acid, and spread on microscope slides to obtainmetaphase chromosomes. The slides were pretreated with 100 μg of RNaseA/ml in 2×SSC (1×SSC is 0.15 M NaCl, 0.015 M sodium citrate) for 1 h at37° C., washed in 2×SSC, and put through an ethanol dehydration series(70, 90, and 100% ethanol). The chromosomes were denatured at 70° C. for5 min in 70% formamide-2×SSC, plunged into ice-cold 70% ethanol, anddehydrated as before. One hundred nanograms of 16RNP-ECFP and 50nanograms of mouse gamma-satellite (Horz et al., Nucl.Acids Res. 9,683-696, 1981) were labelled with digoxigenin-11-dUTP andbiotin-16-dUTP, respectively, by nick translation (Boehringer) followingmanufacturer's instructions. Labelled probes were ethanol precipitatedwith 5 μg of herring sperm DNA and the RNP probe with 1 μg of cot-1 DNA;resuspended in 50% formamide-2×SSC-1% Tween 20-10% dextran sulfate;denatured at 75° C., the RNP probe preannealed for 30 min at 37° C.;pooled and applied to the slides. Hybridization was carried outovernight at 37° C. The slides were washed four times for 3 min eachtime in 50% formamide-2×SSC at 45° C., four times for 3 min each time in2×SSC at 45° C., and four times for 3 min each time in 0.1×SSC at 60° C.After being wahed for 5 min in 4×SSC-0.1% Tween 20, the slides wereblocked for 5 min in 4×SSC-5% low-fat skimmed milk. The biotin wasdetected by 30 min incubation at 37° C. with each of the following:avidin-conjugated Texas Red (Vector Laboratories) followed bybiotynylated anti-avidin (Vector Laboratories) and avidin-conjugatedTexas Red (Vector Laboratories). Digoxigenin was detected at the sametime as biotin with each of the following: anti-digoxigenin-fluorescein(FITC, Boehringer) followed by mouse anti-FITC (DAKO) and horsefluorescein-conjugated anti mouse IgG (Vector Laboratories). Betweenevery two incubations, the slides were washed three times for 2 min eachtime in 4×SSC-0.1% Tween 20. The slides were counterstained with DAPI(4′-6-diamidino-2-phenylindole) and mounted in Vectashield (Vector).Images were examined with an oil ×100 objective on a Olympus BX40fluorescence microscope. The images were captured with a Photometricscooled charge-couple device camera and Vysis Smartcaprture software.

Copy Number Determination

Genomic DNA was prepared from cell clones by standard procedures(Sambrook et al., 1989). Transfected gene copy number was determined bySoutern blot analysis of Hincll digested genomic DNA. The transgene wasdetected as a 2.5 kbp band by hybridization to a 1 kpb fragment from16RNP-EGFP, comprising the neomycin resistance gene, labelled with[α-³²P] dCTP following manufacturer's instructions (Megaprime DNAlabelling system, Amersham). For normalization, blots weresimultaneously hybridized with a 1 kbp Ncol fragment, labelled as above,derived from the murine vav locus (Ogilvy et al., 1998) which gave a 1.4kbp band. As copy number standards, DNA from several pWE-TSN clones wasdigested with Pstl and hybridized to the above probes. Hybridizationsignal quantification was performed with a Cyclone PhorsphorImager(Packard).

Analysis of GFP Expression in Transfected Ltk Clones

The transfected cells were maintained on G418 selection at 200 μg/ml.Cells at 80-100% confluency were stripped off 6-well plates forexpression analysis of GFP. Cells were washed with PBS and incubated inTrypsin/EDTA (Sigma) until they had detached from the surface of theplates. An excess of DMEM (Gibco) supplemented with 10% foetal calfserum (Sigma) was added to the cells and transferred to 5 ml polystyreneround-bottom tubes. The cells were then analyzed on a Becton-DickinsonFACScan for the measurement of GFP fluorescence in comparison to theautofluorescence of an untransfected control.

Production of EBV Receptor Construct

TA DNA fragment containing the cytomegalovirus (CMV) promoter, theenhanced green fluorescent protein (EGFP) and the simian virus 40 (SV40)polyadenylation sequence, was removed from the vector, pEGFP-N1(Clontech), by restriction endonuclease digestion with Ase I and Afl IIusing the manufacturers recommended conditions (NEB). The DNA waselectrophoresed on a 0.5% agarose gel to separate the fragment from thevector backbone. The DNA fragment was cut out of the gel and purifiedfrom the gel slice using the standard glass milk purification technique.The fragment was blunted using T4 DNA polymerase (NEB) according to themanufacturers conditions and purified by 1:1 (v/v) extraction withphenol:chloroform:isoamylalcohol (25:24:1) followed by ethanolprecipitation

The reporter cassette was then cloned into the Epstein-Barr virus (EBV)vector, p220.2 (described in International Patent Application WO98/07876). P220.2 was restriction endonuclease digested with Hind III (aunique site in the multiple cloning sequence (MCS) of the vector),blunted and purified in the same way as described above. The reportercassette was ligated into p220.2 using T4 DNA ligase (Promega). Theligation reaction was performed in a 10 μl volume using 200 ng of thelinearised p220.2 and either a molar equivalent or 5 molar excess of theCMV-EGFP-SV40pA fragment, in 1× ligation buffer (Promega). The reactionwas incubated overnight at room temperature. 2.5 μl of the ligationswere transformed into electrocompetent DH5αE. coli cells byelectroporation at 2.5 kV, 400 Ω, 25 μF followed by the addition of 900μl of SOB medium and incubation at 37° C. for 1 hour. 200 μl of each ofthe transformations were plated on LB-ampicillin agar plates andincubated overnight at 37° C.

The resulting colonies were screened for the presence of the reportercassette by colony polymerase chain reaction (PCR) with DNA primers inthe CMV and EGFP sequence, using Taq polymerase (AdvancedBiotechnologies) with the manufacturers standard conditions. Positivecolonies were grown overnight in LB-ampicillin medium and were analysedas alkaline-lysis DNA minipreparations (Qiagen). The DNAs were screenedfor the correct orientation of the fragment using Bam HI restrictionendonuclease digestion. The resultant construct was named p220.EGFP.

p220.EGFP was demonstrated to express EGFP by analysis on aBecton-Dickinson FACScan, after electroporation into KS62 cells, usingessentially the same method as described below.

Production of EBV Reporter Constructs Containing the hnRNPA2 16 kb(RNP16) UCOE fragment.

A Sal I site was removed from p220.EGFP by partial restrictionendonuclease digestion of the vector with Sal I, followed by bluntingand religation of the vector, thus leaving a unique Sal I site in themultiple cloning site (MCS) of the vector which could be utilised forthe cloning of the 16 kb RNP fragment. The resultant vector wasrestriction endonuclease digested with Sal I, treated with calfintestinal phoshatase (to prevent recircularisation of the vector duringthe ligation) and purified by phenol:chlorofom extraction and ethanolprecipitation.

The 16 kb RNP fragment was removed from the vector, MA551, using therestriction endonuclease, Sal I, and was blunted, purified byelectroelution and ligated into the linearised vector. The ligationreactions were set up in the same way as previously described (using amolar equivalent amount of the fragment), followed by transformation andscreening of the colonies for the presence of the fragments. Colonieswere screened as DNA minipreparations, with positive colonies beingconfirmed by agarose gel electrophoresis analysis. The correctorientation of the 16 kb RNP fragment was determined by restrictionendonuclease analysis using Not I. The resultant construct was namedp220.RNP16.

Transfection of EBV Reporter Constructs into HeLa Cells.

HeLa cells were transfected in 6-well plates with p220.EGFP andp220.RNP16, using the CL22 peptide-mediated delivery system described inInternational Patent Application WO 98/35984 and described below. Afterculture for 24 hours, hygromycin B (Calbiochem) selection was added to afinal concentration of 400 μg/ml. Hygromycin B-resistant colonies ofcells were maintained in culture and analysed periodically for GFPexpression on a Becton-Dickinson FACScan. Cells were routinely splitinto 24-well plates the day before analysis so that they wereapproximately 50% confluent on the day of analysis. For the expressiontime course, a marker region was set which contained the GFP-expressingpopulation of cells and this marker was used to investigate thestability of GFP expression over time. Transfected HeLa cells were alsotaken off hygromycin B selection to investigate the stability of GFPexpression, in the absence/presence of the UCOE, without selectionpressure.

P Cloning of CET200

EGFPN1 was restricted with NheI/NotII and the following oligos wereannealed and inserted to create the multiple cloning site (MCS):

5′ CTAGCGTTCGAAGTTTAAACGC 3″′ (SEQ ID NO:18) 5′ GGCCGCGTTTAAACTTCGAACG3″′ (SEQ ID NO:19)

The resulting plasmid was restricted with AseI blunted and the 8.3 kbHindIII fragment blunted RNP A2 fragment inserted. The resultingorientation was then determined creating the final vector CET200 (seeFIG. 49).

Cloning CET210

pUC19 was restricted with EcoRI/ArI and blunted, removing one PvuI sitethus creating a unique PvuI site for linearisation (pUC19Δ). The MCS wasremoved from pEGFPN1 by digestion with NheI/AgeI and blunted. Thiscreates the NheI site. The CMV EGFP SV40 cassette was removed as aAfIII-blunt AseI fragment and inserted into pUC19-Δ that had beenrestricted with PvuII and pGK puro bGH (from pGK-puro-BKS) was insertedwithNdeI. The resulting vector was then restricted with NheI/NotIremoving EGFP and the MCS inserted as described above. The MCScontaining vector was then restricted with HindIII and the 8.3 kb RNPHindIII fragment inserted creating the final vector CET210 (see FIG.49).

Preparation of Plasmid Containing a UCOE

Cloning of RNP-UCOE Containing Reporter Constructs

p8 kb Hind BKS contained a 8.3 kb HindIII genomic fragment of the RNPlocus contained the promoters and first exons of RNPA2 and HP1H-γ genes.

pCMVEGFP-IRES was constructed by digesting pEGFP-N1 (Clontech, same asCMV-EGFP FIG. 35) with KpnI and NotI to liberate the EGFP sequence, thiswas then ligated into plRESneo (Clontech) that had been partiallydigested with KpnI and then NotI. This created a vector with the EGFPgene 3′ to the CMV promoter and 5′ to IRESneo.

IntronA-CMV was cloned by taking the 1.5 kb IntronA-CMV fragment frompTX0350 (a pUC based CMV IntronA-MAGE1 plasmid) with NruI (blunt cutter)and Hind III. pEGFP-NI was digested with AseI and the ends of thefragment were then end filled with Klenow and T4 DNA Polymerase. Thiswas then digested with HindIII to obtain a 4.2 Kb fragment. Bothfragments were then ligated overnight.

p4.0CMV was constructed by excising a 4 kb fragment from p8 kb Hind BKSwith BamHI/HindIII/BstEII digestion. The ends of the fragment were thenend-filled with Klenow and T4 DNA polymerase.

pEGFP-N1 (Clontech) was linearised with AseI, the ends blunted as aboveand then treated with calf intestinal phosphatase (CIP). Both fragmentswere then ligated overnight. The resultant clones were screened for bothforward and reverse orientations of the 4 kb UCOE insert.

p7.5CMV was constructed by excising the 8.3 kb fragment from p8 kb HindBKS with HindIII digestion. The ends of the fragment were then endfilled with Klenow and T4 DNA Polymerase. pEGFP-NI (Clontech) waslinearised with AseI, the ends were blunted as above and then treatedwith calf intestinal phosphatase (CIP). Both fragments were then ligatedovernight. The resultant clones were screened for both forward andreverse orientations of the 8.3 kb UCOE insert.

p16CMV was constructed by excising a 16 kb fragment from MA551 (hnRNPA2genomic clone containing 5 kb 5′ and 1.5 kb 3′ sequence including theentire coding region) by Sal I digestion. The ends of the fragment werethen end filled with Klenow and T4 DNA Polymerase. pEGFP-NI (Clontech)was linearised with AseI, the ends were blunted as above and thentreated with calf intestinal phosphatase (CIP). Both fragments were thenligated overnight. The resultant clones were screened for both forwardand reverse orientations of the 16 kb UCOE insert.

Transfection of HeLa Cells Using the CL22 Peptide

The CL22 peptide has the amino acid sequence:

NH₂-KKKKKKGGFLGFWRGENGRKTRSAYERMCNILKGK-COOH (SEQ ID NO:20)

The CL22 peptide was used as a transfecting agent in accordance with themethods described in WO 98/35984.

HeLa cells are routinely cultured in EF10 media, splitting a confluentflask 1:10 every 3 to 4 days. 24 Hours prior to transfection, cells wereseeded at 5×10⁴ per well (6 well plate). Complexes were formed 1 hourprior to transfection by mixing equal volumes of DNA:CL22, which are atconcentrations of 40 μg/ml and 80 μg/ml respectively in Hepes bufferedsaline (10 mM Hepes pH 7.4, 150 mM NaCl), and incubated at roomtemperature for 1 hour. Media was removed from cells, which were thenwashed with 1% phosphate buffered saline. 2.5 μg of DNA:complex (125 μl)was then added to the cells and the volume made up to 1 ml with RAQ(RPMI media (Sigma), 0.1% human albumin, 137 μM chloroquine (addedfresh)) which gives a final concentration of chloroquine of 120 μM.Cells and complex were incubated for 5 hours at 37° C. The complex wasthen removed and replaced with EF10 media (Minimal Essential medium(Sigma), 10% Foetal calf serum, 100 unit/ml penicillin/0.1 mg/mlstreptomycin, 1× Non-Essential amino acids (Sigma)).

Analysis of GFP Expression in Transfected HeLa Cells

Cells were stripped off 6-well plates for expression analysis of GFP.Cells were washed with phosphate buffered saline (PBS; Gibco) andincubated in Trypsin/EDTA (Sigma) until they had detached from thesurface of the plates. An excess of EF10 medium (Gibco) supplementedwith 10% foetal calf serum (FCS; Sigma) was added to the cells and thecells transferred to 5 ml polystyrene round-bottom tubes. The cells werethen analysed on a Becton-Dickinson FACScan for the detection of GFPexpression in comparison to the autofluorescence of the parental cellpopulation.

Preparation of Total DNA Samples

In order to examine the episomal DNA content of the transfectedpopulations, a total preparation of cellular DNA was made. The cellswere washed with PBS and then lysed with lysis buffer [10 mM tris pH7.5, 10 mM EDTA pH 8.0, 10 mM NaCl and 0.5% Sarcosyl to which was addedfresh Proteinase K 1 mg/ml F/C]. The cell lysate was scraped off theplate and transferred to an eppendorf tube with a wide bore pipette.Following overnight incubation at 65° C. the cell lysate wasphenol/chloroform extracted and ethanol precipitated. The DNA pellet wasresuspended in TE pH 8.0.

Detection of Episomal DNA in Total Genomic DNA Samples

Total genomic DNAs, prepared from transfected cells, 7 days aftertransfection, were restriction endonuclease digested using anendonuclease that linearised the DNA constructs used in the transfectionand therefore any episomal DNA present in the sample. Apa LI (NEB) wasused for mock, CMV-EGFP, IntronA-CMV and 4.0CMV forward and reversesamples. BspLU11 I (Boehringer) was used for 7.5CMV forward and reversesamples. 10 μl (20% of the sample) of total genomic DNA were digestedwith 30 units of restriction endonuclease, for 16 hours according to themanufacturers recommended conditions. The samples were electrophoresedfor 400 volt/hours on a 0.6% agarose gel along with 100 pg or 4 ng oflinearised plasmid controls. The gel was then transferred to Hybond-NHybridisation transfer membrane (Amersham) by Southern blotting.Briefly, the gel was incubated in 0.25M HCl for 15 minutes to depurinatethe DNA, followed by denaturation in 1.5M NaCl/0.5M NaOH for 45 minutesand neutralisation in 1.5M NaCl/0.5M Tris-CI, pH 7.0, for 45 minutes.The DNA was then transferred from the gel to the membrane by capillaryblotting in 20×SSC (3M NaCl, 0.3M Na₃citrate-2H₂O, pH 7.0) for 16 hours.The filter was air-dried for 1 hour and cross-linked for 2 minutes usinga UVP CL-100 ultraviolet crosslinker (GRI) at an energy setting of 1200.The membrane was probed using a radioactive EGFP probe using “Churchhybridisation conditions”. The membrane was prehybridised in 0.5M NaPipH 7.2, 1% SDS at 65° C. for longer than 2 hours. An EGFP fragment ofDNA was removed from pEGFP-N1 (Clontech) by restriction endonucleasedigestion with Bgl II/Not I (NEB), separated by electrophoresis andpurified from the gel slice using a GFX™ PCR DNA and Gel BandPurification kit (Amersham Pharmacia Biotech). 50 ng of the EGFPfragment were labelled with α-³²P dCTP (3000Ci/mmol; Amersham) using aMegaprime DNA labeling kit (Amersham). The labelled probe was mixed with100 μl of 10 mg/ml salmon sperm DNA, incubated at 95° C. for 10 minutesand placed on ice followed by addition to the hybridisation. Themembrane was hybridised for 16 hours at 65° C., followed by two 30minute washes in 40 mM NaPi pH7.2, 1% SDS at 65° C. The radiolabelledmembrane was then analysed on a Cyclone storage phoshor system (Packard)after exposure on a super resolution phosphor screen.

Fluorescence Microscopy

The transfected cells cultured in 6-well plates were viewed underfluorescence using a Zeiss Axiovert S100 inverted microscope.Photography was carried out at regular timepoints throughout using aZeiss MC100 camera and Fujichrome Provia 400ASA film.

Example 1

Analysis of the Human TBP Gene Locus

Mapping the TBP Gene Domain

The human TBP gene is 20 kb in length (Chalut et al., 1995), located onchromosome 6q27-tel (Heng et al., 1994) and is closely linked to thegene encoding the protein C5 which forms part of a ubiquitous proteosome(FIGS. 1A and C; Trachtulec, Z. et al., 1997). The C5 gene isdivergently transcribed from a position 1 kb upstream from the cap siteof TBP. TBP and C5 may therefore comprise dual promoters. This hasimportant ramifications with regards to the construction of expressionvectors based on TBP since dual promoters do not necessarily functionwith equal efficiency in both directions (see Gavalas and Zalkin, 1995).

Sequence analysis has revealed that the TBP/C5 promoter regions arecontained within a methylation-free, CpG-island of 3.4 kb. This extendsfrom a Fsp I site within intron 1 of C5 and a HindIII site within intron1 of TBP and encompasses the most 5′ 1 kb sequences of the first intronof both genes as well as the 1.4 kb region between their transcriptionalstart sites (FIG. 1B).

The human TBP gene locus consists of 3 closely linked genes. The PSMB1gene (also referred to herein as C5) is divergently transcribed from aposition 1 kb upstream from the cap site of TBP. The 3′ end of arecently identified gene, PDCD2 is located 5 kb downstream of TBP. These3 transcription units span a total of 50 kb. Downstream of the PSMB1gene in the direction of the centromere, there is a region of at least80 kb which consists of blocks of repeat sequence DNA with noidentifiable structural genes. Upstream of the PDCD2 gene toward thetelomere there is a 30 kb stretch of repeat, non-coding sequencesfollowed by a potential new transcription unit. The PDCD2 gene isapproximately 150 kb from the start of the telomeric repeat region. Thismakes the TBP locus the first structural gene cluster from the telomereon the long arm of chromsome 6. Pa

Pattern of Gene Expression from the TBP Domain

The tissue distribution of expression from within the TBP gene clusterwas assessed using a commercially available dot-blot prepared withpoly(A)⁺-RNA derived from a wide range of human tissues and cell types(FIG. 35A). Hybridisation of this dot-blot with appropriate probesshowed that the PSMB1 (FIG. 35B), PDCD2 (FIG. 35C) and TBP (FIG. 35D)genes are all ubiquitously expressed. These data confirm that the TBPlocus consists exclusively of a ubiquitously expressed chromatin domain.

Mapping Transgene Integrity in Mice Harbouring pCP2-TLN

The pCYPAC-2 derived clone pCP2-TLN (FIG. 1) which is 90 kb in lengthwas used to generate transgenic mice. This clone starts at a position 46kb downstream of the C5 gene (65 kb 5′ of TBP) and terminates 4.5 kb 3′of TBP. This clone therefore possesses both C5 and TBP genes in theirentirety.

Three transgenic lines with pCP2-TLN have been produced. The initialSouthern blot analysis with probes derived from the ends of pCP2-TLNshowed that line TLN:3 possesses two copies of the transgene (FIGS. 2a,b lanes TLN-3) in a head-to-tail configuration (FIG. 3 a, lanesTLN:3). However, one copy appears to have suffered a 5′ deletion, whichextends into the TBP promoter (FIG. 4, lanes TLN:3). Line TLN:8 by endfragment analysis appeared to harbour 3 copies of pCP2-TLN (FIG. 2 a,blanes TLN-8). Line TLN:28 appeared to harbour several copies at multipleintegration sites (FIG. 3 a, lanes TLN:28).

A summary of the initial analysis of transgene copy number and integrityin these TLN mice is shown in FIG. 3B.

Further analysis of the transgenic lines produced with pCP2-TLN has nowshown that line TLN:3 contains two deleted copies of pCP2-TLN such thata single functional copy of the TBP and PSMB1 genes remains intact (FIG.3C, TLN:3). Line TLN:8 harbours two, tandem integrated copies ofpCP2-TLN (FIG. 3C, TLN:8). Line TLN:28 possesses 4 tandem arrangedcopies of pCP2-TLN (FIG. 4, TLN:28). The deletions at the 5′ and 3′ endsof the transgene tandem arrays in TLN:8 and TLN:28 still leave the PSMB1and TBP genes intact.

As expected the methylation-free island of TBP/C5 is preserved intransgenic mice (data not shown) as has been observed for the 5′ regionof other genes which harbour a CpG-rich domain (e.g. murine Thy-1;Kolsto et al., 1986)

Expression Analysis of the TBP and C5 Transgenes on pCP2-TLN in Mice

An RT-PCR based assay that would simultaneously detect both theendogenous murine as well as the human transgene TBP and C5 message wasdeveloped. Primers (TB-14 and TB-22) for the RT-PCR reactions wereselected from a region of homology between the human and mouse TBP cDNAsequence (FIG. 5 b). This allows an RT-PCR product of 284 bp to beproduced from both mRNAs by a single pair of primers. In order todistinguish between the human and mouse TBP products, minor basedifferences resulting in changes in the presence of restriction enzymesites are exploited. Digestion with Bsp 14071 cleaves the human PCRproduct, giving rise to a fragment of 221 nucleotides (nt) (FIG. 6 a).Similarly, from a region of homology between the human and mouse C5 cDNAsequence (FIG. 5 a), allowed the generation of an RT-PCR product of350nt from both sequences. Cleavage with PstI reduced the size of theproduct derived from the murine C5 mRNA to 173nt (FIG. 7 a)

Primers TB14 (FIG. 5 b) and C5RTF (FIG. 5 a) were end-labelled with ³²Presulting in the generation of radioactive products after the PCRreaction. These products are finally resolved by electrophoresis ondenaturing polyacrylamide gels (FIGS. 6 b-c and 7 b).

Total RNA (1 μg) from various tissues of transgenic mouse lines TLN:3,TLN:8, and TLN:28, were subjected to the above analytical procedure andquantified by PhosphorImager analysis (FIG. 8). All mice showedsignificant levels of expression of both the human TBP and C5 transgenesin all tissues analysed including TLN:3, which harbours a single intactcopy of these two genes. Most importantly, a reproducible level ofexpression was observed between tissues in a given mouse line especiallyfor C5. This indicates that the TLN clone in all likelihood possesses aubiquitous chromatin opening capability. However, some variation in thelevel of expression per transgene copy number was observed between mouselines. In addition, expression of TBP in line TLN:8 between tissues alsovaried from 5-40%. These results suggest that although TLN possesses achromatin opening capability, the C5 and especially the TBP promotersare prone to positive and negative transcriptional interference. This inturn implies that the inherent transcriptional activating potential ofthe TBP and C5 regions on this clone are weak and therefore unable toalways exert a dominant effect over position effects. This is incontrast to what seems to be a chromatin opening UCOE effect of thisregion, which is strong and appears to over-ride such positon effects.This hypothesis is supported by the observation that the weaker TBPpromoter is more prone to variability; compare, for example, the ratioof TBP levels between spleen and muscle with that for C5 in line TLN:8(FIG. 8).

Transgene expression analysis as described previously, was carried outusing tissues from mice that were between 2 and 6 months of age. Thestability of transgene expression was also assessed in 23 month old micefrom lines TLN:3 and TLN:8 by analysing PSMB1 mRNA. Similar results wereobtained in both lines compared to that obtained with the youngeranimals. The result further demonstrates that the transgenes aremaintaining a transcriptionally competent open chromatin structure.

T Expression Analysis of a 40 kb Subclone of the TBP Locus.

The reproducible, physiological levels of expression given by thepCP2-TLN clone in transgenic mice indicate that it possesses aubiquitous chromatin opening capability. As a first step to fine mappingthe region(s) of DNA responsible for this activity, we have begun toanalyse a 40 kb subclone (pCP2-TSN; FIG. 1 a) of the human TBP locus.The pCP2-TSN clone possesses 12 kb of both 5′ and 3′ flanking sequencessurrounding the TBP gene. As a result it only harbours a complete TBPand a 3′ truncated mutant of C5.

Previous work with the human β-globin LCR demonstrated that an initialindication for the presence of LCR activity may be obtained by comparingexpression levels between stable transfected tissue culture cell clonesharbouring a single copy of the transgene. It has been found that themore complete the LCR element, the higher the degree of reproducibilityof expression between independent clones. Expression analysis ofpCP2-TSN was conducted using this strategy to assess for the presence ofLCR-type activity.

pCP2-TSN was first cloned into the cosmid vector pWE15 (Clontech) whichpossesses a neomycin resistance gene (FIG. 9). The resulting pWE-TBPconstruct was then used to generate stable transfected clones of murinefibroblast L-cells. The transgene copy number of 23 clones was thendetermined by Southern blot analysis (FIG. 10). A number of clonesrepresenting a range of copy numbers were then selected and analysed fortransgene expression as described for the transgenic mice above. Theresults are summarised in FIG. 11 and show that expression at or abovephysiological levels are obtained per copy of the transgene up to anumber of eight. With copy numbers of 20 or more, expression levels pertransgene are reduced to 30-40% of wild type.

These data demonstrate that reproducible, physiological levels ofexpression can be produced by pCP2-TSN at both single and multipletransgene copy numbers. This strongly suggests that this genomic clonepossess a ubiquitous chromatin opening capability. There are clearly anumber of clones (e.g. number 4, 33 and 6), which show a pronounced“ositive” position effect giving rise to expression levels that aremarkedly greater than physiological per transgene copy. This would bethe anticipated outcome in certain cases where integration of thetransgene had taken place within already open, active chromatin. Thenearby presence of a strong transcriptional enhancer under thesecircumstances would be expected to have a stimulatory effect on theinherently weak TBP promoter.

The stability of expression of the constructs was tested over a 60 dayperiod. Expression levels were found to remain constant (FIG. 36). Thiswas even the case when drug selective pressure was removed (FIG. 36,lanes marked BG418). In addition, expression remained stable throughsuccessive freeze and thaw cycles of the cells regardless of whetherdrug selective pressure was maintained.

Expression analysis of a 25 kb sub-clone of the TBP locus

The 25 kb genomic clone (TPO) spanning the TBP gene with 1 kb 5′ and 5kb 3′ flanking sequences (FIG. 1C) was cloned into the polylinker regionof a modified pBluescript vector harbouring a puromycin resistance geneto give pBL-TPO-puro as described above. The construct was used togenerate stable transfected clones of murine fibroblast L-cells.

The pBL-TPO-puro construct gave similar results to those obtained usingthe TSN construct (FIG. 37). The data demonstrate that reproduciblephysiological levels of expression can be produced by both TSN and TPOat single and multiple transgene copy numbers. The data is consistentwith the genomic clones possessing a ubiquitous chromatin openingcapability. This surmise is further enhanced by the finding that TPOclone numbers 7 (two copies), 29 (single copy) and 34 (two copies) arecentromeric integration events (data shown below) demonstrating that thegenomic fragment has the ability to express from within aheterochromatin environment.

There are clearly a number of clones (e.g. FIG. 37, clone 11), whichshow a pronounced “positive” position effect giving rise to expressionlevels that are greater than physiological per transgene copy. Thiswould be the anticipated outcome in certain cases where integration ofthe transgene had taken place within already open, active chromatin. Thenearby presence of a strong transcriptional enhancer under thesecircumstances would be expected to have a stimulatory effect on theinherently weak TBP promoter.

Similar results have also been obtained using HeLa cells instead of CHOcells (data not shown).

Mapping DNase I Hypersensitive Sites All

All known LCR elements have been found to be regions of high,tissue-specific DNase I hypersensitivity, indicative of the highly openchromatin configuration which these elements are thought to generate. Wehave therefore begun to analyse for the presence of DNase Ihypersensitive (HS) sites both within and around the human TBP gene.FIG. 12 summaries a series of experiments using nuclei from the humanmyelogenous leukaemia cell line K562, which maps DNase I HS sites over a40 kb region starting from 12 kb 5′ and extending 4.5 kb 3′ of the TBPgene. The only HS sites that are evident throughout this region map tothe immediate promoter regions of the C5 and TBP genes (FIG. 12, toppanel, HindIII digest/HindIII-Xba I probe). These HS sites correlatewell to previously identified promoter elements important for TBP and C5gene expression as determined by transient transfection assays (Tumara,T. et al., 1994; Foulds and Hawley, 1997). However, it would appear thatif LCR-type elements are present within this locus, they are at aconsiderable distance from the transcriptional start sites of both theTBP and C5 genes. This places any LCR-type element outside of the 40 kbclone spanning the TBP gene that has given an initial indication ofubiquitous chromatin opening capability.

FISH Analysis

A total of 34 clones carrying 1-2 copies of the human TBP transgene wereanalyzed by FISH. The TBP transgene and the heterochromatin component ofthe mouse centromere, the gamma or major satellite, were detected withFluorescein and Texas Red, respectively. This produced green and redfluorescent signals in the clones in which the transgene had integratedinto the chromosome arm (see FIG. 39A). However, in the case ofcentromeric integration both signals co-localized and a mixture of bothcolours could be detected as a yellow fluorescent signal. Two clones,344-6 and 344-37, out of the 18 generated with pWE-TSN, showed thetransgenic signal in the centromeric region. In clone 344-6, the TBPtransgene had integrated in the centromere of a Robertsonian chromosome,whereas integration in clone 344-37 was in a typical mouse acrocentricchromosome.

Three clones, 440-7, 440-29, and 440-34, out of the 16 generated withpBL3-TPO-puro, showed centromeric integration in typical acrocentricchromosomes. Clone 440-29, which carried a single copy of the TBPtransgene, showed the TBP signal clearly surrounded by heterochromaticsatellite sequences (see FIGS. 39B and C). It was further shown thatthese clones continued to express TBP at physiological levels for atleast 12 to 14 weeks in the absence of selection (data not shown).

These results show that a single copy of the 25 kb fragment of the TBPlocus (TPO) is capable of ensuring physiological expression even in thecontext of a heterochromatic location (i.e. centromeric integration),and thus provides formal proof of chromatin opening (Sabbattini P,Georgiou A, Sinclair C, Dillon N (1999) Analysis of mice with single andmultiple copies of transgenes reveals a novel arrangement for theλ5-V_(preB1) locus control region. Molecular and Cellular Biology 19:671B679).

Example 2

Analysis of the Human hnRNP A2 Gene Locus Mapping the hnRNP A2 GeneDomain

The hnRNP A2 gene is composed of 12 exons spanning 10 kb and is highlyhomologous to the hnRNP-A1 gene in its coding sequence and overallintron/exon structure indicating that it may have arisen by geneduplication (Biamonti et al., 1994). However, unlike the A1 gene noA2-specific pseudogenes have been found (Burd et al., 1989; Biamonti etal., 1994). In addition, the A1 and A2 genes are not genetically linkedbeing on human chromosomes 12q13.1 (Saccone et al., 1992) and 7p15(Biamonti et al., 1994) respectively. FIG. 13A depicts a genetic map ofthe human hnRNP A2 locus present on the 160 kb pCYPAC-2 derived cloneMA160. This genomic fragment possesses 110 kb 5′ and 50 kb of 3′flanking sequences. The DNA sequence of the 4.5 kb region upstream ofthe known transcriptional start site of the hnRNP-A2 was determined.This identified the position of the gene for theheterochromatin-associated protein HP1 γ to be divergently transcribedfrom a position approximately 1-2 kb 5′ of the hnRNP-A2 cap site (FIG.13C). Southern blot analysis indicates that the entire HP1 γ gene iscontained within a region of 10 kb (data not shown).

Therefore the TBP and hnRNP-A2 gene loci share the common feature ofclosely linked, divergently transcribed promotors.

The pattern of expression of the HP1 HP1 γ ene within human tissues wasassessed on a dot-blot prepared with poly(A)⁺-RNA derived from a widerange of human tissues and cell types. The results (FIG. 38) show thatthe gene, like that for hnRNP-A2 is also ubiquitously expressed. The twogenes can therefore be seen to form a ubiquitously expressed gene domainsimilar to that of the TBP locus.

Functional Analysis of the hnRNP A2 Locus in Transgenic Mice

MA160 (FIG. 13A) was used to generate transgenic mice. Southern blotanalysis of the two founders that have bred through to the F1 stage hasshown that these lines possess 1-2 copies of the transgene (data notshown).

A similar RT-PCR based assay to that used for TBP was used to analyseexpression of the human hnRNP A2 transgene. The cDNA sequence of themurine hnRNP A2 is not known. Therefore, we could not select a region ofhomology between human and mouse hnRNP A2 by sequence comparison forRT-PCR amplification. We initially chose two primers Hn9 and Hn11, whichcorrespond to sequences within exons 10 and 12 respectively of humanhnRNP A2 (FIG. 14A) and gives rise to an RT-PCR product of 270 bp.However, we found that these two primers gave an identical sized productfrom both human and mouse RNA preparations (FIG. 14B) indicating aregion of homology between these two species. Tests with a range ofrestriction enzymes also revealed that Hind III is able to cut themurine (FIG. 14B, lane HindIII M) but not the human (FIG. 14B, laneHindIII H) product to give a fragment of 170 bp.

Total RNA (1 μg) prepared from various tissues of an F1 transgenic miceof line Hn35 and Hn55, were then analysed using the above method with³²P-end labelled 5′Hn9 (FIG. 16). PhosphorImager analysis was used toquantify the ratio of human to mouse RT-PCR products. The results (FIG.17A) show that reproducible, physiological levels of expression pertransgene copy number are obtained in all tissue types analysed.

Analysis of 60 kb Subclone of the hnRNP A2 Locus in Transgenic Mice

The data obtained with the MA160 pCYPAC-derived clone indicate that thisgenomic fragment possesses a ubiquitous chromatin opening capability. Inorder to further define the location of the DNA region(s) responsiblefor this activity, transgenic mice were generated with a 60 kb Aat IIsub-fragment (Aa60) obtained from MA160 (FIG. 13B). This fragmentpossesses 30 kb 5′ and 20 kb 3′ flanking sequences around the hnRNPA-2gene.

Three transgenic mice (Aa7, Aa23 and Aa31) have been generated to datewith the Aa60 fragment, two of which (Aa23 and 31) have bred through toestablish lines. Estimated transgene copy numbers are: Aa7, 3; Aa23,1-2; Aa31, 1-2).

Total RNA (1 μg) from a range of tissues was analysed for transgeneexpression as described above. The results are shown in FIG. 15 andquantified by PhosphorImager (FIG. 17B). These data show that alltransgenic mice express at a reproducible level per transgene copynumber in all tissues analysed. This indicated that the ubiquitouschromatin opening capacity shown by MA160 is preserved on the Aa60sub-fragment.

Mapping of DNase I Hypersensitive Sites

The results of preliminary experiments to map DNase I HS sites over a20-25 kb region 5′ of the transcriptional start point of the human hnRNPA2 gene are shown in FIG. 18. A 766 bp probe from exon 2 on a doublerestriction enzyme digest with Aat II and Cla I, gave a series of threeHS sites (FIG. 18, upper panel) corresponding to positions −1.1, −0.7and −0.1 kb 5′ of the hnRNP A2 gene (FIG. 18, lower panel). We have alsoextended the analysis to 12-13 kb downstream of the transcriptionalstart of hnRNP-A2 and no further HS sites where identified.

As in the case of the TBP/C5 locus, these HS sites correspond to the 1-2kb region between the promoter of hnRNP A2 and the HP1HHP1H-γ e. NoLCR-type HS sites were detected indicating that the chromatin openingcapacity of this locus is not associated with this type of element.

The data presented clearly show we have been able to obtainreproducible, ubiquitous, physiological levels of expression with twodifferent gene loci (TBP and hnRNP A2) in all tissues of transgenicmice. This indicates that genetic control elements, not derived from anLCR, with a ubiquitous chromatin opening capability do indeed exist.

It is important to note that the data herein presented demonstrate atotally different function to the previously published results usingpromoter-enhancer combinations from other ubiquitously expressed genessuch as human β-actin (e.g. see Ray, P. et al., 1991; Yamashita et al.,1993; Deprimo et al., 1996), murine hydroxy-methylglutaryl CoA reductase(Mehtali et al., 1990), murine adenosine deaminase (Winston et al., 1992and 1996), human ornithine decarboxylase (Halmekyt ö et al., 1991) andmurine phosphoglycerate kinase-1 (McBurney et al., 1994). In theseearlier studies high levels of expression were observed in only a subsetof tissues and a chromatin opening function was not demonstrated ortested for.

In the case of the TBP gene, expression data from tissue culture cells(FIG. 11) indicate that this ubiquitous chromatin opening capacity iscontained within a 40 kb genomic fragment with 12 kb of 5′ and 3′flanking sequences (pCP2-TSN, FIG. 1 a).

Transgenic mouse data with a 60 kb fragment spanning the hnRNP A2 gene(Aa60; FIG. 13B), indicate that the region with a ubiquitous chromatinopening capacity is contained on this fragment (FIGS. 15-17).

The only DNase I HS sites that have been mapped to these regions to datecorrespond to classical promoter rather than LCR-type elements.Therefore, the regions of DNA which act as ubiquitous chromatin openingelements (UCOEs) do not meet the definition of LCR elements which areassociated with genes that are expressed in a tissue-specific orrestricted manner. UCOEs and their activities can therefore clearly bedistinguished from LCRs and LCR derived elements.

Expression Vector Development

Sub-fragments of the 60 kb RNP region are assayed for UCOE activityusing reporter based assays.

Expression vectors containing sub-fragments located in the dual promoterregion between RNP and HP1H-γ were designed using both GFP and a Neo^(R)reporter genes, as described above and as shown in FIG. 22. Theseinclude a control vector with the RNP promoter driving GFP/Neoexpression (RNP), a vector comprising the 5.5 kb fragment upstream ofthe RNP promoter region and the RNP promoter (5.5RNP), vectorsconstructed using a splice acceptor strategy wherein the spliceacceptor/branch consensus sequences (derived from exon 2 of the RNPgene) were cloned in front of the GFP gene (ensuring that the entire CpGisland including sequences from RNP intron 1 can be tested in the samereporter-based assay), resulting in exon 1/part of intron 1 upstream ofGFP (7.5RNP), carrying 7.5 kb of the RNP gene preceeding the GFP gene,and a vector comprising the 1.5 kb fragment upstream of the RNP promoterregion and the RNP promoter (1.5RNP).

Expression vectors comprising the heterologous promoter CMV are alsodescribed above and are shown in FIG. 23. These include control vectorswith the CMV promoter driving GFP/Neo expression with an internalribosome binding site (CMV-EGFP-IRES) and without an internal bindingsite (CMV-EGFP), a vector comprising the 5.5 kb fragment upstream of theRNP promoter region and the CMV promoter driving GFP/Neo expression(5.5CMV), a vector comprising 4.0 kb sequence encompassing the RNP andthe HP1 HHP1H-γ romoters and the CMV promoter driving GFP/Neo expression(4.0CMV), and a vector comprising 7.5 kb sequences of the RNP geneincluding exon 1 and part of intron 1, and the CMV promoter drivingGFP-Neo expression.

These constructs were transfected into CHO cells by electroporation, asdescribed above. Addition of the 5.5 kb region in front of the RNPpromoter resulted in a 3.5-fold increase in number of G418^(R) colonies,FIG. 24. Transfection of these same constructs into COS7 cells using anucleic acid condensing peptide delivery strategy showed an increase incolony numbers closer to 7-fold (data not shown).

A 1.5-fold increase in colony numbers was also observed aftertransfection of the CMV-based vectors (i.e. CMV vs. 5.5CMV) into CHOcells, FIG. 24.

Ring cloning of colonies from these transfections resulted in stableG418^(R) cell lines which could then be analysed for GFP expressionlevels. The FACS data is shown in FIG. 25. Addition of the upstreamsequences resulted in a 3.5-fold increase in GFP expression when assayedwith the endogenous promoter (RNP vs 5.5 RNP). An increase in GFPexpression is also seen with addition of the 5.5 kb sequence in front ofthe heterologous CMV promoter (CMV vs 5.5CMV).

Extension of the constructs to include the entire methylation freeisland showed no increase in the number of G418^(R) colonies as comparedwith 5.5RNP, but there was an increase in the average median GFPfluorescence (5.5RNP cf. 7.5RNP; see FIG. 26).

GFP expression of individual clones and restricted pools (approx. 100colonies) were followed over time culturing the cells with/without G418selection. Clones generated with the RNP promoter alone showed dramaticinstability, with the percentage of GFP expressing cells rapidlydecreasing over time. Clones expressing GFP from the 5.5RNP construct incomparison were stable for more than 3 months. Although CMV-GFP poolsinitially show better stability, after prolonged culturing in theabsence of G418 a decrease in the number of GFP expressing cells wasevident, in comparison to the 5.5CMV populations which remainedcompletely stable. FIGS. 27 and 28 show FACS profiles of thesepopulations clearly indicating a shift to the left i.e. an increasingproportion of non-fluorescent cells with the CMV-GFP construct. Incontrast the 5.5CMV-GFP pools show a stable uniform peak of expressionover time.

The percentage of low or non-expressing cells is estimated from a gatedpopulation M1.

The studies on the RNP locus have narrowed in on a 5.5 kb regioncovering the dual promoters of the RNP and HP1H-γ genes. Extension ofthis fragment in the 3′ direction (7.5RNP or 8.5RNP) shows anenhancement in the level of gene expression and may relate tomaintaining the methylation free islands intact. It has also been foundthat minimisation of the 5.5 kb sequences to a 1.5 kb region (1.5RNP,FIG. 23) does not dramatically affect the outcome of reportertransfection studies, in terms of both the numbers of G418R colonies andexpression as determined by FACsSanalysis (FIG. 29). However, 1.5RNPdoes not confer the stability of gene expression as shown by 5.5RNP and7.5RNP. FIG. 30 shows the percentage of GFP expressing cells rapidlyreduces over 68 days.

The construct 4.0CMV was designed so that the entire 4 kb of sequencerepresenting the CpG methylation free island remained intact. Inaddition, the cassette was inserted in front of CMV-EGFP (4.0CMV-EGFP-F(forward) and 4.0CMV-EGFP-R (reverse)) in both orientations. FIG. 31shows a dramatic enhancement (greater than 10-fold) of GFP medianfluorescence, as compared to the standard CMV-GFP construct, CMV-EGFP.It is also shown that this boost of GFP expression occurs when the 4 kbcassette is in both the forward and reverse orientations.

In terms of stability of gene expression, the vectors containing theupstream 5.5 kb RNP sequences when transfected into CHO cells andfollowed over time show a definite advantage. Most importantly thisstability is not only limited to the endogenous promoter but alsoconfers a stability advantage to the heterologous and widely used CMVpromoter.

FIG. 32 shows CMV based constructs 4.0CMV and 7.5CMV with control vectorCMV-EGFP transfected into CHO cells and analysed at day 13post-transfection following G418 selection. A substantial increase(15-20 fold) in median fluorescence can be seen by adding the 4.0 or the7.5 kb fragments from the RNP locus in front of the CMV promoter. Thisincrease was independent of the orientation of the fragment (data notshown).

FIG. 33 shows the percentage of GFP expressing cells in the same G418selected pools as in FIG. 32. It can be seen that inclusion of the 4.0and the 7.5 kb fragments enhances the percentage of GFP positive cellsin the G418 selected population. In addition, the populations appearrelatively stable over time, although from previous experiments it wasevident that CMV-EGFP instability is only apparent after approximately60 days in culture.

FIG. 34 shows colony numbers after transfection of CHO cells withequivalent molar amounts of various constructs. The 7.5CMV constructsshow approximately 2.5-fold more colonies than the control vectorCMV-EGFP. These observations are consistent with 7.5CMV-F ensuring anenhanced number of productive integration events and therefore withthere being a chromatin opening/maintaining capacity to the 7.5 kbfragment.

Adenovirus Vector Containing a UCOE

At the present time adenovirus (Ad) is the vector system giving the mostefficient delivery of genes to many cell types of interest for genetherapy. Many of the most promising gene therapies in clinicaldevelopment use this vector system, notably vectors derived from Adsubtype 5. The utility of Ad for human gene therapy could besubstantially increased by improving expression of the therapeutic genesin two main ways. The first involves increasing the level of transgeneexpression in order to obtain the maximum effect with the minimum dose,and this applies whichever promoter is used. The second involvesimproving tissue specific or tumour-specific promoters, such that theyretain specificity but give stronger expression in the permissive cells.Although several promoters giving good specificity for particulartissues or tumour types are known, the level of expression they give inthe permissive cells is generally too weak to be of real therapeuticbenefit. An example of this is the promoter of the mousealpha-foetoprotein (AFP) gene, which gives expression that is weak butvery specific for hepatoma (liver cancer) cells (Bui et al, 1997, HumanGene Therapy, 8, 2173-2182). Such tumour-specific promoters are ofparticular interest for Gene-Directed Enzyme Prodrug Therapy (GDEPT) forcancer, which exploits gene delivery to accomplish targetedchemotherapy. In GDEPT a gene encoding a prodrug converting enzyme isdelivered to tumour cells, for example by injecting the delivery vectorinto tumours. Subsequent administration of a relatively harmless prodrugconverts this into a potent cytotoxic drug which kills the cellsexpressing the enzyme in situ. An example concerns the enzymenitroreductase (NTR) and the prodrug CB1954 (Bridgewater et al, 1995,Eur. J. Cancer, 31A, 2362-2370). Adenovirus vectors give the mostefficient delivery of genes encoding such enzymes, for example by directinjection into tumours.

Construction of an Ad Expressing NTR from the AFP Promoter and a UCOE

A recombinant type 5 adenovirus vector was made which expresses the NTRgene from the AFP promoter preceded by the 4 kb RNP UCOE (the sequenceof FIG. 20 between nucleotides 4102 and 8286). The 4 kb UCOE was firstcloned as a Pme1 fragment into pTX0379, an intermediate vector whichcarries the NTR gene preceded by the AFP promoter (Bui et al, 1997,Human Gene Therapy, 8, 2173-2182) and flanked by Ad5 sequences (1-359,3525-10589), by blunt end ligation into the Cla1 site located 5′ to theAFP promoter. Restriction digestion was used to confirm the presence ofa single UCOE copy and to establish the orientation of the UCOE. Arecombinant Ad construct was then generated using the plasmid pTXO384which contains the UCOE fragment in reverse orientation and the Adpackaging cell line Per.C6, which was developed and supplied byIntrogene (Fallaux et al, 1998, Human Gene Therapy, 9, 1909-1917). Theprocedure supplied by Introgene was used for viral rescue. EssentiallypTXO384 was linearised with Swa1 and co-transfected into Per.C6 cellswith Swa1-linearised backbone vector pPS1160, which carries the rightend of Ad5 and a region of overlap with pTXO384 such that a recombinantAd is generated by homologous recombination. Virus produced byhomologous recombination in the transfected cells was pooled anddesignated CTL208.

NTR Expression in Cell Lines in Vitro

Larger scale virus preparations were made using standard procedures forCTL208, and two other recombinant Ad viruses. These were CTL203, whichcarries the NTR gene preceded by the AFP promoter and minimal enhancerbut no UCOE fragment, and CTL102 which carries the NTR gene preceded bythe CMV promoter. The CMV promoter is commonly used in recombinant Advectors to give strong expression in a wide range of tissue and tumourtypes. CTL203 and CTL102 share the same Ad5 backbone as CTL208 and wereidentical to it except in the elements used for transcription of the NTRgene.

CTL203, 208 and 102 were then used to transduce two cell lines in vitroto investigate the level and specificity of NTR expression. These werethe primary human hepatoma cell line HepG2 which expresses AFP, andKLN205, a mouse squamous cell carcinoma line which does not express AFP.Exponentially growing cells were harvested from tissue culture plates bybrief trypsinisation, resuspended in infection medium at 1.25×10⁴ viablecells/ml and plated into 6 well plates. The viruses were added to thewells before attachment at a multiplicity of 50, and for CTL203 atmultiplicities of 100 and 500 also. After 90 mins the foetal calf serumconcentration was adjusted to 10% and the cells incubated for a total of24 hours. Cell lysates were made from the infected cells by hypotoniclysis, then cell debris cleared by centrifugation in eppendorf tubes. AnELISA was performed to quantify the NTR protein in the supernatants.This involved coating Nunc-Immuno Maxisorp Assay Plates with recombinantNTR, adding 50 μl of each hypotonic lysate per well in duplicate andincubating overnight at 4° C. The samples were then washed 3× with 0.5%Tween in PBS and incubated with a sheep anti-NTR polyclonal antiserum(100 μl per well of a 1 in 2000 dilution in PBS/Tween for 30 mins atroom temperature. After washing off excess primary antibodyHRP-conjugated secondary antibody was applied, this being donkeyanti-sheep (100 μl per well of 1 in 5000 in PBS/Tween). After a further30 min incubation the samples were washed with PBS before developmentwith 100 μl per well of TMB substrate (1 ml TMB solution, 1 mg/ml inDMSO+9 ml of 0.05M phosphate-citrate buffer+2 μl of 30% v/v H₂O₂ per 10ml) for 10 mins at room temperature. The reactions were stopped byaddition of 25 μl of 2M H₂SO₄ per well and read at 450 nm using a platereader.

FIG. 46 shows the results of these ELISAs. It shows that CTL203, withNTR expressed from the AFP promoter/enhancer, gave weak but specific NTRexpression, detectable only in the AFP positive cell line. CTL102 (withNTR expressed from the CMV promoter) gave much higher and non-specificexpression, with very similar levels of NTR in both cell lines.Strikingly, AFP positive HepG2 cells infected with CTL208 (UCOE+AFPpromoter driving expression of NTR) expressed NTR at a higher level thenCTL102 infected cells, whereas CTL208 infected AFP negative KLN205 cellsexpressed significantly less NTR than those infected with CTL102. Thesedata show that the UCOE dramatically enhances expression in the contextof Ad, with partial retention of specificity.

NTR Expression and Anti-Tumour Effects In Vivo

Tumour-specific promoters are preferable to non-specific promoters forcancer gene therapy from the safety viewpoint, because they will givelower expression of the transgene in normal tissues. This isparticularly important for Ad-based gene therapies because afterinjection into tumours some of the virus tends to escape from the tumourand following systemic dissemination tends to transduce normal tissues.In particular Ad gives very efficient transduction of liver cells, suchthat liver damage is usually the dose-limiting toxicity for Ad genetherapies. In the case of GDEPT the use of strong promoters able to giveexpression in normal tissues, such as the CMV promoter, can lead tokilling of normal liver cells expressing NTR. This problem canpotentially be avoided or minimised using tumour-specific promoters,which would be advantageous providing these give sufficiently strongexpression in the tumour cells to give anti-tumour effects. CTL208 wastherefore compared to CTL102 for NTR gene expression in tumour cells andliver cells following injection into tumours in mice, and foranti-tumour effects. The congenitally athymic nude mouse strain BALB/cnu/nu was used. The mice were males free of specifc pathogens, agedeight to twelve weeks at the commencement of the experiments, andmaintained in microisolator cages equipped with filter tops.Exponentially growing HepG2 cells cultured in vitro were used as tumourinocula. The cells were cultured in shake flasks, harvested bytrypsinisation and centrifugation for 5 min at 800 g, washed andresuspended in sterile saline solution. Cell viability was estimated bytrypan blue dye exclusion, and only single cell suspensions of greaterthan 90% viability were used. Mice were injected sub-cutaneously in theflank with 2-5×10⁶ cells, under general anaesthesia, induced byintraperitoneal injection of 0.2 ml of a xylizine (Chanelle AnimalHealth Ltd, Liverpool, UK) and ketamine (Willows Francis Veterinary,Crawley, UK) mixture at a concentration of 1 mg/ml and 10 mg/mlrespectively. In the first experiment CTL102 or CTL208 were injectedinto sub-cutaneous HepG2 tumours of size 25-60 mm² (size expressed assurface area determined by multiplying the longest diameter with itsgreatest perpendicular diameter, length×width′mm²) growing in nude mice.Single doses of 7.5×10⁹ particles were used for each virus. The animalswere sacrificed 48 hours later, their tumours and livers excised, fixedin buffered 4% formalin/PBS for 24 hours and processed forparaffin-embedding and sectioning using standard protocols. Serial 3 msections were cut and immunostained to detect cells expressing NTR byindirect immunoperoxidase staining using a sheep anti-NTR antiserum(Polyclonal Antibodies Ltd) and VECTASTAIN Elite ABC kit (Vector Labs).These histological sections were examined using standard microscopicequipment and the percentage of cells expressing NTR in the entirelivers and tumours were estimated by microscopy. FIG. 47 shows theresults for each mouse. It demonstrates that the UCOE in combinationwith the (otherwise weak) AFP promoter gives strong NTR expression inAFP positive tumours in mice, such that on average CTL208 gives verysimilar numbers of tumour cells expressing NTR at detectable levels asCTL102 following injection into tumours. Intra-tumoral injection ofCTL102, however, led to NTR expression detectable in the liver for 5 outof 6 animals for CTL102, but 0 out of 6 for CTL208. This result confirmsthat in CTL208 the UCOE-AFP promoter combination gives expression in AFPpositive tumour cells similar to or stronger than the CMV promoter, butshows much less expression in (AFP negative) normal tissues.

To confirm that the UCOE elevates expression from the AFP promoter totherapeutically useful levels CTL208 and CTL102 were compared for theirability to confer anti-tumour effects in combination with the prodrugCB1954. Nude mice bearing sub-cutaneous HepG2 tumours of size 25 to 60mm² were given single injections of CTL102 or CTL208, at doses of either7.5×10⁹ or 2×10¹⁰ particles. 24 hours later CB1954 administration to themice commenced. CB1954 (Oxford Asymmetry, Oxford, UK) was dissolved inDMSO (Sigma, St Louis, Mo., USA) to give a concentration of 20 mg/ml.Immediately prior to dosing this solution was diluted 1:5 in sterilesaline solution to give a final concentration of 4 mg/ml. Mice receivedfive equal daily doses intraperitoneally without anaesthesia. For acontrol group of mice the tumours were injected with PBS instead ofvirus 24 hours before commencing prodrug administration. Tumour size wasmeasured daily using vernier calipers for the next 27 days. FIG. 48shows the results. For the control group given CB1954 and neither virus,7/7 tumours continued to grow rapidly. Tumour regressions were observedin some of the mice in all the groups given both NTR expressing virusand CB1954. With CTL102 regressions were observed in ⅜ mice given thelower dose, and {fraction (4/8)} mice given the higher dose. With CTL208regressions were observed in ⅝ and {fraction (6/8)} mice respectively.These results confirm that, in CTL208, the UCOE elevates NTR expressionfrom the AFP promoter in permissive tumour cells to levels which exceedthose given by the strong CMV promoter and this results in a superioranti-tumour effect in a mouse model of the clinical situation for GDEPT.

These results demonstrate two important and useful properties of theUCOE. First, it substantially improves expression in the context of Ad,a non-integrating vector of great potential in gene therapy. Second, itelevates expression from weak but specific promoters to much more usefullevels with retention of useful specificity.

FISH Analysis

Copy number was determined in 31 16 RNP-EGFP clones in mouse Ltk cells.Due to the low amount of DNA used in the transfection (0.5-1.0 μg), thepercentage of single copy clones was very high (83%). Moreover, EGFPexpression varied more than two-fold within the single copy clones,indicating that the transgene was susceptible to positive and negativeposition effects. Nonetheless, three single copy clones had integratedin centromeric heterochromatin (FIG. 42), indicating that this constructis able to open chromatin. Clones F1 and G6 showed the 16RNP-EGFPtransgene had integrated in one of the centromeres of metacentricchromosomes originated by Robertsonian translocations (FIGS. B, C),whereas in clone 13, integration had occurred in the centromere of atypical mouse acrocentric chromosome (FIG. D).

Expression of Erythropoietin (EPO) In Vectors CET300 and CET301

Construction of EPO Expression Vectors CET300 and CET301

The erythropoietin (EPO) coding sequence was amplified by polymerasechain reaction (PCR) from a human fetal liver Quick-Clone™ cDNA library(Clontech, Palo Alto, US.) using primers EP2 (5′-CAGGTCGCTGAGGGAC-3′)(SEQ ID NO:21) and EP4 (5′-CTCGACGGGGTTCAGG-3′) (SEQ ID NO:22). Theresulting 705 bp product, which included the entire open reading frame,was subcloned into the vector pCR3.1 using the Eukaryotic TA cloning kit(Invitrogen, Groningen, The Netherlands), to create the vector pCR-EPO.The EPO sequence was verified by automated DNA sequencing on bothstrands. A 790 bp NheI-Eco RV fragment, containing the EPO codingsequence, was excised from pCR-EPO and subcloned between the Nhe I andPme I sites of the vectors CET200 and CET210 (containing the 7.5 kb RNPfragments in the forward and reverse orientations respectively), togenerate the vectors CET300 and CET301 respectively. A control vector,pCMV-EPO, was generated by excising the EGFP coding sequence frompEGFP-N1 as a NheI-NotI fragment and replacing it with a NheI-Not Ifragment from pCR-EPO containing the EPO coding sequence.

Expression of Erythropoietin in CHO Cells

Plasmids CET300, CET301 and pCMV-EPO were linearised using therestriction endonuclease Dra III. Restricted DNA was then purified byextraction with phenol-chloroform followed by ethanol precipitation. DNAwas resuspended in sterile water and equimolar amounts of the plasmidswere electroporated into CHO cells. Viable cells were plated in 225 cm³culture flasks and stable transfected cells were selected by replacingthe medium after 24 hrs for complete medium containing 0.6 mg/ml G418.Cells were grown in this medium until G418-resistant colonies werepresent (about 10 days after electroporation). The flasks were thenstripped and cells were seeded at 10⁶ cells/well in a 6 well dishcontaining 1 ml of complete medium. After 48 hrs the medium was removedand the levels of erythropoietin in the media were quantitated by enzymelinked immunosorbent assay (ELISA) using a Quantikine^(R) IVD^(R) HumanEPO immunoassay kit (R & D systems, Minneapolis, US). The levels of EPOproduced by the constructs CET300, CET301 and pCMV-EPO were 1780 U/ml,1040 U/ml and 128 U/ml respectively (FIG. 40). Therefore, constructsCET300 and CET301, containing the 7.5 kb RNP fragment in forward andreverse orientations, produced EPO in the above experiment at levelsapproximately 14-fold and 8-fold higher, respectively, than the controlplasmid pCMV-EPO which contains the strong ubiquitous CMV promoter todrive expression of EPO.

GFP expression in Hela cells transfected with EBV reporter constructswith or without the 16 kb UCOE fragment of hnRNPA2

In the initial experiment with cells maintained on hygromycin selection,the RNP16 UCOE-containing construct (p220.RNP16) gave high level,homogeneous expression of EGFP by day 23, whereas a more heterogeneouspattern of EGFP expression was observed with p220.EGFP (constructwithout the UCOE). EGFP expression in the p220.EGFP-transfected poolswas gradually lost, whereas expression remained stable for 160 days withthe p220.RNP16-transfected pools.

Three repeat experiments demonstrated the same pattern of high level,homogeneous EGFP expression in p220.RNP16-transfected pools, withheterogeneous expression again observed in the p220.EGFP-transfectedpools. As with the initial experiment, the expression of EGFP was stablewith the RNP16 UCOE and was unstable without the UCOE, with expressiondropping dramatically by 30-40 days (FIG. 43).

A further experiment was performed wherein hygromycin selection wasremoved at day 27. The results show that even without selection EGFPexpression is stable with the RNP16 UCOE and was unstable without theUCOE (FIG. 44).

Example 3

Plasmid Containing a UCOE

FIG. 50 shows the constructs generated and fragments used in comparisonto the hnRNPA2 endogenous genomic locus.

FIG. 51 shows a graph of the FACS analysis with median fluorescence ofthe transiently transfected HeLa populations. The cells were transfectedusing the CL22 peptide condensed reporter plasmids as indicated above.It can be seen that the duration of expression of the control CMV-GFPreporter construct is short-lived and dramatically decreases from 24 to48 hours post-transfection.

In contrast to the control, the UCOE containing plasmid 7.5CMV-Fcontinues to show significant GFP expression over an extended period oftime, at least 9 days post-transfection. In repeat experiments GFPexpression can be seen at 14 days post-transfection.

FIG. 52 shows representative low magnification field of views of thetransiently transfected HeLa cell populations. The data correlates withthe FACS analyses and enables the cells to be visibly followed over asimilar time-course. At 24 hours post-transfection significant numbersof GFP positive cells are visible in both the control CMV-GFP and 7.5CMVtransient populations (FIGS. 52A and B). In fact it can be seen that at24 hours there were more GFP positive cells in the control populationthan in the 7.5CMV transfected population. This is due to the fact thatthe quantity of input DNA in both cases was not gene dosage corrected,resulting in significantly more copies of the control plasmid pertransfection. However, at 6 days post-transfection there were very fewif any positive fluorescent cells left in the CMV-EGFP controlpopulation (FIG. 52C). In contrast 6 days post-transfection the 7.5CMVtransfected HeLa cells continued to show significant numbers of GFPexpressing cells (FIG. 52D). In fact even 14 days after transfectionpositively fluorescing cells could easily be detected (data not shown).

Total DNA was recovered from various time points throughout theexperiment, linearised, run on a gel and blotted (see Materials andMethods). Interestingly at day 6 even in the control population of cellswhere little or no expression of GFP was detected, the plasmid could bereadily detected in an unintegrated state (data not shown). This wouldsuggest that the rapid loss in gene expression seen with the CMV-GFPcontrol plasmid is not due to chronic loss of the plasmid template butrather to a mechanism of chromatin shut-down of gene expression.

Transient Transfection of CHO cells with Erythropoietin ExpressionVectors

Supercoiled forms of plasmids CET300, CET301 and CMV-EPO wereelectroporated into CHO cells using standard conditions (975 μF, 250V).Viable cells were then seeded at 10⁶ cells in a 6-well dish containing 1ml of complete CHO medium. The medium was then removed at 24 hrintervals and replaced with 1 ml of fresh medium. Media samples werecollected in this fashion for 9 days and erythropoietin levels were thenquantitated by ELISA using a Quantikine^(R) IVD^(R) Human EPOimmunoassay kit (R & D systems, Minneapolis, US). The attached figureshows a time course of erythropoietin expression by cells transfectedwith CET300, CET301 and CMV-EPO plasmids. Erythropoietin expressioncontinued to rise for 48 hrs in all cell populations. Thereafter,erythropoietin expression by cells transfected with CMV-EPO fell on adaily basis. Whereas, levels of EPO expression by cells transfected withCET300 or CET301 continued to rise throughout the 9-day period (FIG.45).

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1. A method for obtaining a desired gene product comprising culturing ahost cell comprising a vector comprising a polynucleotide comprising: a.an element comprising an extended methylation-free CpG-island; b. anexpressible gene, wherein said expressible gene is operably-linked tosaid CpG-island; and c. a promoter, operably-linked to said gene,wherein said promoter is not naturally operably-linked to saidCpG-island, wherein said element facilitates reproducible activation oftranscription of said gene in two or more tissue types.
 2. A method forobtaining a desired gene product comprising culturing a host cellcomprising a vector comprising a polynucleotide comprising: a. anelement comprising an extended methylation-free CpG-island comprising atleast one endogenous promoter; b. an expressible gene, wherein saidexpressible gene is operably-linked to said CpG-island, further whereinsaid expressible gene is not naturally linked to said CpG-island; and c.a further promoter, external to said CpG-island, operably-linked to saidgene, wherein said further promoter is not naturally operably-linked tosaid CpG-island, wherein said element facilitates reproducibleactivation of transcription of said gene in two or more tissue types. 3.A method for obtaining a desired gene product comprising culturing ahost cell comprising a vector comprising a polynucleotide comprising: a.an element comprising an extended methylation-free CpG-island comprisingat least one endogenous promoter; b. an expressible gene, wherein saidexpressible gene is operably-linked to said CpG-island, further whereinsaid expressible gene is not naturally linked to said CpG-island; and c.a further promoter, external to said CpG-island, operably-linked to saidgene, wherein said further promoter is not naturally operably-linked tosaid CpG-island, wherein said element facilitates reproducibleactivation of transcription of said gene.
 4. A method for reproduciblyexpressing a gene comprising operably-linked said gene to apolynucleotide comprising: a. an element comprising an extendedmethylation-free CpG-island; b. an expressible gene, wherein saidexpressible gene is operably-linked to said CpG-island; and c. apromoter, operably-linked to said gene, wherein said promoter is notnaturally operably-linked to said CpG-island, wherein said elementfacilitates reproducible activation of transcription of said gene in twoor more tissue types.
 5. A method for reproducibly expressing a genecomprising operably-linked said gene to said polynucleotide comprising:a. an element comprising an extended methylation-free CpG-islandcomprising at least one endugenous promoter; b. an expressible gene,wherein said expressible gene is operably-linked to said CpG-island,further wherein said expressible gene is not naturally linked to saidCpG-island; and c. a further promoter, external to said CpG-island,operably-linked to said gene, wherein said further promoter is notnaturally operably-linked to said CpG-island, wherein said elementfacilitates reproducible activation of transcription of said gene in twoor more tissue types.
 6. A method for reproducibly expressing a genecomprising operably-linked said gene to a polynucleotide comprising: a.an element comprising an extended methylation-free CpG-island comprisingat least one endogenous promoter; b. an expressible gene, wherein saidexpressible gene is operably-linked to said CpG-island, further whereinsaid expressible gene is not naturally linked to said CpG-island; and c.a further promoter, external to said CpG-island, operably-linked to saidgene, wherein said further promoter is not naturally operably-linked tosaid CpG-island, wherein said element facilitates reproducibleactivation of transcription of said gene.
 7. A method for preparing avector for reproducibly expressing a gene comprising operably-linked apolynucleotide comprising: a. an element comprising an extendedmethylation-free CpG-island; b. an expressible gene, wherein saidexpressible gene is operably-linked to said CpG-island; and c. apromoter, operably-linked to said gene, wherein said promoter is notnaturally operably-linked to said CpG-island, wherein said elementfacilitates reproducible activation of transcription of said gene in twoor more tissue types.
 8. A method for preparing a vector forreproducibly expressing a gene comprising operably-linked apolynucleotide comprising: a. an element comprising an extendedmethylation-free CpG-island comprising at least one endogenous promoter;b. an expressible gene, wherein said expressible gene is operably-linkedto said CpG-island, further wherein said expressible gene is notnaturally linked to said CpG-island; and c. a further promoter, externalto said CpG-island, operably-linked to said gene, wherein said furtherpromoter is not naturally operably-linked to said CpG-island, whereinsaid element facilitates reproducible activation of transcription ofsaid gene in two or more tissue types.
 9. A method for preparing avector for reproducibly expressing a gene comprising operably-linked toa polynucleotide comprising: a. an element comprising an extendedmethylation-free CpG-island comprising at least one endogenous promoter;b. an expressible gene, wherein said expressible gene is operably-linkedto said CpG-island, further wherein said expressible gene is notnaturally linked to said CpG-island; and c. a further promoter, externalto said CpG-island, operably-linked to said gene, wherein said furtherpromoter is not naturally operably-linked to said CpG-island, whereinsaid element facilitates reproducible activation of transcription ofsaid gene.